Tissue slice selection method, and embedded block preparing cassette

The present invention is to provide a tissue slice selection method and an embedded block-preparing cassette which are capable of suppressing occurrence of errors in inspection results. The present invention makes it possible to suppress the occurrence of errors in inspection results by including: an embedded block-preparing process of preparing an embedded block by embedding both a biological tissue fragment and a standard substance in the same embedding agent; an embedded tissue slice preparing step of slicing the embedded block to prepare a sheet-like embedded tissue slice having a tissue slice and standard substance slices appearing on a surface thereof; and a tissue slice selecting step of selecting the tissue slice to be inspected, based on a light signal from the standard substance slice.

TECHNICAL FIELD

The present invention relates to a tissue section selection method and an embedded block-preparing cassette.

BACKGROUND ART

It is known that in a cancer tissue, specific genes are amplified and proteins, the products of the genes, are overexpressed. In the inspection of cancer and the like, over-expression of the protein or gene amplification in a specimen obtained from a living body is detected by an immunohistochemical staining method, and the condition of the specimen is inspected.

For example, HER2/neu protein, which is a gene product of an HER2/neu gene, is over-expressed with a high frequency in tumor cells such as breast cancer, the HER2/neu gene being one of oncogenes. Therefore, it is possible to inspect whether a subject suffers from cancer by detecting the HER2/neu protein in tumor cells using the immunohistochemical staining method and determining the presence or absence of over-expression of the HER2/neu protein.

As an inspection kit to which such principle is applied, commercially available is an immunohistochemical staining kit using an anti-human HER2/neu gene product monoclonal antibody (SV2-61γ) (animal species: mouse) that targets the HER2/neu protein expressed in the tumor cells and recognizes an extracellular region of the HER2/neu protein.

In such immunohistochemical staining kit, a specimen is immunohistochemically stained (hereinafter, simply referred to as stained) through the following steps. First, for example, a specimen is prepared in a stainable format through a provision step of providing an embedded block in which a biological tissue fragment collected from a human is embedded in an embedding agent such as paraffin, a preparation step of slicing the embedded block to a desired thickness using a microtome to prepare a sheet-like embedded tissue section in which a periphery of the tissue section is surrounded by an embedding agent section, and a placing step of placing the embedded tissue section prepared in this manner on a slide glass.

Subsequently, a deparaffinization treatment step of removing paraffin from the embedded tissue section on the slide glass and an antigen retrieval treatment step of activating the tissue section are performed.

Finally, performed are a blocking treatment step such as an endogenous peroxidase treatment with 3 V/V % peroxide, a primary antibody reaction treatment step in which a primary antibody is bound to a specific antigen in the tissue section, a secondary antibody reaction treatment step in which a secondary antibody bound to a labeled enzyme is bound to the primary antibody, and a staining treatment step in which the specific antigen in the tissue section is stained by using chromogenic development with a substrate solution containing a chromogenic substrate (for example, DAB: 3,3′-diaminobenzidine). The tissue section is stained through such a series of steps (NTL 1).

At this time, a control slide for determining suitability of the staining is provided separately from the embedded tissue section, and the tissue section and the control slide are stained at the same time. The control slide has a specimen sample, and thus can determine the suitability of the staining from the staining intensity of the specimen sample. If it is confirmed that the staining has been suitably performed from the control slide, a staining intensity score of the tissue section is determined based on predetermined criteria, and from the result of the score determination (evaluation), the presence or absence of a lesion tissue in the tissue section is determined (NTL 2).

CITATION LIST

Non Patent Literature

SUMMARY OF INVENTION

Technical Problem

A tissue section for inspection using such immunohistochemical staining is usually prepared by slicing an embedded block to a thickness of several micrometers using an instrument such as a microtome, as described above. In the slicing operation using the microtome, a variance in thickness may occur depending on a skill level of the engineer, a preparation environment (for example, room temperature and humidity), and a type of embedded tissue, and the manipulation deviation may occur even by the same engineer and thus a variance in thickness may occur.

In the inspection using the immunohistochemical staining, since the staining intensity changes depending on the thickness of the tissue section, variance in the thickness of the tissue section has a large influence on the inspection result. For example, when a variance occurs in the thickness of multiple tissue sections used for the inspection, the tissue sections will not exhibit the same staining intensity and will have a different staining intensity depending on the thickness of the tissue section even when the staining is simultaneously performed under the same conditions. Therefore, when there is a variance in thickness of the tissue sections, a score which is different from the state of the actual tissue section may be determined, which may cause an error in the inspection result.

In the inspection using the immunohistochemical staining, furthermore, suitability of the staining is determined using a control slide provided separately from the embedded tissue section to improve the accuracy of the inspection result. However, even in this case, there is a demand for improving the accuracy of the inspection result by suppressing occurrence of an error in the inspection result.

Furthermore, before the embedded block is prepared, a fixing treatment, for example, is performed, in which the biological tissue fragment is immersed in a fixing solution such as a formalin aqueous solution for a predetermined period of time to appropriately fix the tissue form and/or the antigen activity of the biological tissue fragment. However, when the fixing time is not appropriate during the fixing treatment and/or the biological tissue fragment is not appropriately fixed, the tissue form and/or the antigen activity of the biological tissue fragment may not be maintained, and thereafter, an error may occur in the inspection result during the inspection of the tissue section.

The present invention has been made in view of the above problems, and an object thereof is to provide a tissue section selection method and an embedded block-preparing cassette which can suppress occurrence of an error in the inspection result.

Solution to Problem

A tissue section selection method of the present invention is a tissue section selection method of selecting a tissue section to be inspected by specifically detecting a marker, which comprises: an embedded block-preparing step for preparing an embedded block by embedding both a biological tissue fragment and a standard substance in the same embedding agent; an embedded tissue section-preparing step of slicing the embedded block for preparing a sheet-like embedded tissue section having the tissue section and a standard substance section appearing on a surface thereof; and a tissue section selecting step for selecting the tissue section to be inspected, based on a light signal from the standard substance section. In the present application, the light signal means an optically detectable signal, for example, a reflected light from the surface of the standard substance section.

The embedded block-preparing cassette of the present invention is an embedded block-preparing cassette used for preparing an embedded block in which a biological tissue fragment is embedded, and includes a box body that is placed on a tray into which a liquid embedding agent is poured. The box body includes: a frame having a through hole which penetrates an upper surface and a lower surface in a region facing the biological tissue fragment on the bottom of the tray; and a standard substance-positioning portion that is provided to face the bottom of the tray and has a positioning hole into which a standard substance is inserted, wherein the standard substance-positioning portion allows the standard substance to be positioned such that the standard substance is present within a height ranging from a lower end to an upper end of the biological tissue fragment when the bottom of the tray is taken as a reference.

A tissue section selection method of the present invention is a tissue section selection method of selecting a tissue section to be inspected by specifically detecting a marker, which comprises: an embedded block-preparing step of, after immersing a biological tissue fragment in a fixing solution for a predetermined period of time, preparing an embedded block by embedding the biological tissue fragment in an embedding agent; an embedded tissue section-preparing step of slicing the embedded block to prepare a sheet-like embedded tissue section having the tissue section appearing on a surface thereof; and a fixing state determining step of performing a staining treatment on the embedded tissue section and determining a fixing state of the tissue section fixed with the fixing solution based on a light signal from an endogenous protein expressed in cells in the tissue section.

Advantageous Effect of Invention

In the tissue section selection method of the present invention, the tissue section to be inspected is selected based on the light signal from the standard substance section, using the sheet-like embedded tissue section in which the tissue section and the standard substance section appear on the same surface. Thus, the inspection can be performed only on the tissue section optimal for the inspection in the tissue section selection method, and thus it is possible to suppress occurrence of an error in the inspection result.

In the embedded block-preparing cassette of the present invention, since the embedded block is prepared by embedding both the biological tissue fragment and the standard substance in the same embedding agent, it is possible to prepare an embedded tissue section in which the tissue section and the standard substance section appear on the same surface when the embedded block is sliced. Thus, in the embedded block-preparing cassette, the tissue section to be inspected can be selected based on a light signal from the standard substance section, and inspection can be performed only on the tissue section optimal for the inspection. Thus, it is possible to suppress occurrence of an error in the inspection result.

Since the tissue section selection method of the present invention enables evaluation of the fixing state of the tissue section fixed with the fixing solution, inspection can be performed only on the tissue section in a fixing state optimal for the inspection, and occurrence of an error in the inspection result can be suppressed.

DESCRIPTION OF EMBODIMENTS

(1) Outline of Embedded Block-preparing Cassette of Present Invention

As illustrated inFIG.1, an embedded block-preparing cassette1according to an embodiment of the present invention is used in a state of being placed on a tray30, and is a tool for embedding both a standard substance21and a biological tissue fragment22in the same embedding agent to prepare an embedded block (which will be described later). The embedding agent (not illustrated) is a material such as paraffin that liquefies when heated and solidifies again when cooled. The embedded block-preparing cassette1is mounted on a slicing instrument, such as a microtome, when the embedded block is sliced. Therefore, the shape and size of the embedded block-preparing cassette1are appropriately selected according to the slicing instrument to be used.

Hereinafter, an embedded block-preparing cassette1will be described by way of a case where the embedded block used for an immunohistochemical staining method is prepared as an example. Further, explained herein is a case in which HER2/neu protein is used as a marker contained in a biological tissue and an immunohistochemical staining method for specifically detecting the HER2/neu protein to inspect a state of a living body is performed.

The embedded block-preparing cassette1is made of a synthetic resin material, such as plastic, phenolic resin, melamine resin, or polystyrene, and includes a box body2having an outer shape formed to conform to a mounting part of the slicing instrument. A frame3is formed in the box body2so as to surround an opening4that penetrates from an upper surface3ato a lower surface. In the case of this embodiment, the frame3is formed into a truncated pyramidal shape in which one side surface of a rectangular parallelepiped is inclined, and the opening4is formed into a quadrilateral shape.

A standard substance-positioning portion5and a through hole6are provided in a region in the opening4surrounded by the frame3. When the embedded block-preparing cassette1is placed on the tray30, the standard substance-positioning portion5is provided in a predetermined region of the opening4so as to keep away from the biological tissue fragment22placed on a bottom surface34of the tray30(hereinafter, simply referred to as a tray bottom) and face the tray bottom34. The standard substance-positioning portion5is provided with a plurality of positioning holes5a, to which standard substances21(which will be described later) are respectively inserted. When the embedded block-preparing cassette1is placed on the tray30, the standard substance-positioning portion5causes the standard substances21to be located so as to be kept away from the biological tissue fragment22placed on the tray bottom34. Accordingly, the standard substances21are disposed to be aligned with the biological tissue fragment22on the tray bottom34by the standard substance-positioning portion5.

A through hole6is provided in the remaining region other than the region in which the standard-positioning portion5is provided in the opening4. The through hole6penetrates the upper surface3aand the lower surface, and when the embedded block-preparing cassette1is placed on the tray30, the biological tissue fragment22placed on the tray bottom34can be exposed through the through hole6when seen from above.

The tray30includes an accommodating portion31that is recessed in a tray shape, a shoulder32that is provided along an outer circumference of the accommodating portion31, and a pair of first flange portion35and second flange portion36provided around the outer circumference of the shoulder32. In the accommodating portion31, the biological tissue fragment22is placed on the tray bottom34in the accommodating portion31when an embedded block is to be prepared. In addition, the accommodating portion31accommodates a liquid embedding agent poured into the tray30after the embedded block-preparing cassette1is placed on the tray30. The liquid embedding agent is cooled and solidified in the accommodating portion31in a state where the embedded block-preparing cassette1is placed on the tray30.

The shoulder32abuts on the embedded block-preparing cassette1and supports the embedded block-preparing cassette1. The first flange portion35and the second flange portion36are respectively provided on the two opposing sides of the shoulder32, and guide the embedded block-preparing cassette1such that the embedded block-preparing cassette1is placed on the shoulder32.

The standard substance21is formed in the same shape (columnar shape) as a hole shape (here, columnar shape) of each positioning hole5ain the standard substance-positioning portion5. The standard substance21refers to a thickness standard substance21athat serves as a reference for the thickness of a tissue section which will be described later, and marker standard substances21b,21c,21dand21ethat serve as a reference for the suitability of the marker detection results, and here, the thickness standard substance21aand the marker standard substances21b,21c,21dand21eare collectively referred to as the standard substance21. In the present embodiment, as described above, a case where the HER2/neu protein in the biological tissue is detected is taken as an example. An amount of HER2/neu protein in the living body is usually evaluated in four stages: 0 (negative), 1+, 2+, and 3+.

For this purpose, as a marker standard substance that serves as a reference for the suitability of the marker detection results, used are a marker standard substance21bmade from a cell line MDA-MB-231 which shows negative staining result, a marker standard substance21cmade from a cell line MDA-MB-175VII showing a staining result of 1+, a marker standard substance21dmade from a cell line MDA-MB-453 showing a staining result of 2+, and a marker standard substance21emade from a cell line SK-BR-3 showing a staining result of 3+.

The marker standard substances21b,21c,21dand21erespectively are prepared by preparing control cell embedded blocks in which the above-described cell lines are embedded, and hollowing out the control cell embedded blocks into a columnar shape using BIOPSY PUNCH (manufactured by Kai Industries).

The thickness standard substance21ais made of a material of which color tone changes as an optical signal depending on the thickness. For example, a pigment-containing urethane foam prepared by mixing a pigment of a predetermined color, such as blue, can be used as a thickness standard substance21a. The thickness standard substance21ais not particularly limited as long as the color information, such as the color tone, changes depending on the thickness.

In the present embodiment, the thickness standard substance21ais prepared by the following procedures. First, a blue pigment-containing urethane foam having a predetermined size (for example, 100 to 110 mm in length, 60 to 70 mm in width, 30 to 40 mm in height, and 35 to 45 g in weight) is prepared. The blue pigment-containing urethane foam is cut in a height direction with a width of approximately 5 mm such that a cutting surface is parallel to a lateral direction of the blue pigment-containing urethane foam. Subsequently, from the blue pigment-containing urethane foam cut into a sheet shape, the vicinity of the center where the number of bubbles contained in the blue pigment-containing urethane foam is small is excised, and the excised foam is further cut into a sheet shape with a thickness of approximately 5 mm, to prepare small urethane foam fragments.

The small urethane foam fragments are placed in a commercially available paraffin-embedded block-preparing cassette EB-W (manufactured by Olympus), and an immobilization treatment is performed using a closed type automatic immobilization embedding apparatus Tissue-Tek VIP (manufactured by Sakura Finetek Japan). The thickness standard substance21ais prepared by hollowing the small urethane foam fragments to which the immobilization treatment was performed and cut into a columnar shape by using tissue micro-array apparatus KIN-2 type (manufactured by Azumaya Medical Instruments Co., Ltd.).

(2) Detailed Configuration of Embedded Block-Preparing Cassette

Here, in addition toFIG.1, the embedded block-preparing cassette1will be described in more detail with reference toFIGS.2and3in which parts that correspond to those ofFIG.1are given the same reference numerals. In the frame3provided in the box body2, a square bar-like horizontal bar9is bridged between a pair of opposing inner walls3bamong the inner walls3bthat surround the inside of the opening4. The horizontal bar9is provided on the same surface as a lower surface3fof the frame3and divides the inside of the opening4into two regions, one region being used as a region for forming the standard substance-positioning portion5, and the other region being used as a region for forming the through hole6.

The standard substance-positioning portion5is formed of the same material as the frame3and has a plurality of positioning hole-forming portions5f,5g,5h,5iand5jhaving a cylindrical shape. The positioning hole-forming portions5f,5g,5h,5iand5jare separated away with a predetermined distance. The adjacent positioning hole-forming portions5f,5gand5h(5h,5i, and5j) are connected to each other by a square bar-like connecting portion5m, and the positioning hole-forming portions5f,5hand5jadjacent to the inner wall3bof the frame3are respectively connected to the inner wall3bby the square bar-like connecting portion5m. The side surfaces of the positioning hole-forming portions5gand5iadjacent to the horizontal bar9are also connected to the horizontal bar9. In the standard substance-positioning portion5, the region other than the positioning hole-forming portions5f,5g,5h,5iand5jand the connecting portion5mis an opening5kthat penetrates the upper surface3aand the lower surface3f.

In this embodiment, the positioning hole-forming portions5f,5g,5h,5iand5jhave the same configuration, and pillar-like hollow regions that penetrate from the upper surface3ato the lower surface3fserve as a positioning hole5a, and the standard substance21can be inserted into the positioning hole5a. In the present embodiment, since the thickness standard substance21aand the marker standard substances21b,21c,21dand21eare used as described above, the standard substance-positioning portion5is provided with five positioning holes5a.

In the standard substance-positioning portion5, the positioning hole-forming portions5f,5g,5h,5iand5jare fixed to the inner wall3bof the frame3via the connecting portion5mor the horizontal bar9, the opening5kis formed among the positioning hole-forming portions5f,5g,5h,5iand5j, and a liquid embedding agent can be poured into the tray bottom34from the opening5k. Further, the horizontal bar9between the through hole6and the standard substance-positioning portion5is provided on the same surface as the lower surface3fof the frame3. Thus, when the embedded block-preparing cassette1is placed on the tray30, a gap can be formed between the tray bottom34and the horizontal bar9, and the liquid embedding agent poured into the tray30may also flow between the region of the through hole6and the region of the standard substance-positioning portion5.

Here, since all of the positioning hole-forming portions5f,5g,5h,5iand5jhave the same configuration, for example, the following description will be given by focusing on the positioning hole-forming portion5g. The positioning hole-forming portion5gis fixed to the inner wall3bof the frame3with the connecting portion5mor the horizontal bar9interposed therebetween such that the positioning hole5afaces the tray bottom34. The positioning hole forming portion5gis formed at a predetermined height, and when the standard substance21is inserted into the positioning hole5a, the one end of the standard substance21is brought into contact with the tray bottom34, and the longitudinal direction of the standard substance21can be maintained to face the vertical direction with respect to the tray bottom34. Accordingly, the positioning hole-forming portion5gcan position the standard substance21in a height range from a lower end to an upper end of the biological tissue fragment22with respect to the tray bottom34when the embedded block is prepared.

In the present embodiment, when the embedded block-preparing cassette1is placed on the tray30on which the biological tissue fragment22is placed on the tray bottom34, the box body2is formed such that the entire biological tissue fragment22is exposed to the outside from the through hole6. Accordingly, in a state where the embedded block-preparing cassette1is placed on the tray30with the biological tissue fragment22placed on the tray bottom34, the biological tissue fragment22is moved on the tray bottom34through the through hole6, and the position of the biological tissue fragment22can be adjusted or the biological tissue fragment22lifted up from the tray bottom34can be pressed against the tray bottom34when the liquid embedding agent is poured.

In the present embodiment, mentioned is a case where the size of the through hole6is larger than the biological tissue fragment22so that the entire biological tissue fragment22placed on the tray bottom34can be seen from above through the through hole6. However, the present invention is not limited thereto. Examples of through holes according to another embodiment may have such size that the entire part between the opposing both end portions of the biological tissue fragment22can be seen from above through the through hole6or may have such size that ⅓ or more of the biological tissue fragment22can be seen from above through the through hole6.

In addition to such a configuration, the box body2is provided with a plate frame portion13disposed in a quadrilateral shape on the lower surface3fof the frame3as illustrated inFIG.3. The plate frame portion13is provided such that an inner wall surface14asurrounds the opening4of the frame3. In other words, the plate frame portion13is provided so as to surround the formation region of the standard substance-positioning portion5and the formation region of the through hole6by the inner wall surface14a.

When the box body2of the embedded block-preparing cassette1is placed on the shoulder32of the tray30, the tip end14of the plate frame portion13abuts the bottom surface34of the tray30, and a space surrounded by the tray bottom34and the inner wall surface14acan be formed. Accordingly, when the embedded block-preparing cassette1is placed on the tray30and the liquid embedding agent is poured from the opening4to prepare an embedded block, the liquid embedding agent accumulates in the space surrounded by the tray bottom34and the plate frame portion13, and the liquid embedding agent poured into the space flows between the region of the through hole6and the region of the standard substance-positioning portion5. Thus, an embedded block in which both the standard substance21and the biological tissue fragment22are embedded in the same embedding agent can be prepared.

(3) How to Use Embedded Block-Preparing Cassette1

Next, a usage of the embedded block-preparing cassette1will be described. The biological tissue fragment22obtained from a living body is placed at a predetermined position on the tray bottom34(mounting step). Next, the embedded block-preparing cassette1is placed on the tray30such that the lower surface3fof the frame portion3of the embedded block-preparing cassette1is placed on the shoulder32of the tray30. Thereby, the biological tissue fragment22on the tray bottom34is disposed in the region of the through hole6in the box body2of the embedded block-preparing cassette1.

Subsequently, the standard substances21are inserted into each positioning hole5aof the standard substance-positioning portion5one by one. At this time, the standard substances21are inserted into the positioning hole5auntil one end abuts on the tray bottom34, and are respectively supported by the positioning hole-forming portions5f,5g,5h,5iand5j. The standard substance21is placed up to the height of the biological tissue fragment22. In the present embodiment, the thickness standard substance21ais inserted into the positioning hole5aof the positioning hole-forming portion5f, and the marker standard substances21b,21c,21d, and21eare respectively inserted into each positioning hole5aof the remaining positioning hole-forming portions5g,5h,5i, and5j(positioning step). Incidentally, the biological tissue fragment22may be placed on the tray bottom34from the through hole6after the embedded block-preparing cassette1is placed on the tray30, or the biological tissue fragment22may be placed on the tray bottom34after the standard substance21is inserted into the positioning hole5a.

In the present embodiment, the outer shape of the standard substance21is columnar in accordance with the columnar shape of the positioning hole5a, and further, the diameter of the standard substance21is selected to be slightly smaller than the diameter of the positioning hole5a, and thus, the standard substance21can be reliably inserted into the positioning hole5a, and the standard substance21can be supported in the positioning hole5a.

Next, a liquid embedding agent, such as paraffin, that is liquefied by heating the embedding agent is poured from the opening4to the tray30(liquid embedding agent-introducing step). The liquid embedding agent is poured until the biological tissue fragment22on the tray bottom34and the standard substance positioning-portion5of the embedded block-preparing cassette1are covered. At this time, the liquid embedding agent accumulates in the space surrounded by the plate frame portion13of the embedded block-preparing cassette1on the tray bottom34, and flows between the region of the through hole6and the region of the standard substance-positioning portion5of the block-preparing cassette1. In addition, the liquid embedding agent accumulates in the gaps in each positioning hole5ainto which the standard substances21are inserted in the positioning hole-forming portions5f,5g,5h,5iand5jor accumulates around the standard substances21exposed from the inside of the positioning holes5a, and the embedding agent covers all of the biological tissue fragment22, the standard substance-positioning portion5and the standard substance21.

When the liquid embedding agent is solidified, the standard substance21is supported by the positioning hole, and thus, for example, the standard substance can be positioned such that the standard substance is present over the entire height range from the lower end to the upper end of the biological tissue fragment22with respect to the tray bottom34. Here, when the liquid embedding agent is poured onto the tray30from the opening4of the embedded block-preparing cassette1, the biological tissue fragment22on the tray bottom34may float above the tray bottom34, or the position of the biological tissue fragment22may also be shifted. In this case, tweezers or the like can be put into the accommodating portion31of the tray30through the through hole6of the frame portion3and the biological tissue fragment22on the tray bottom34can be pressed against the tray bottom34. Thus, the position of the biological tissue fragment22can be adjusted with respect to the height range of the existing standard substance21.

Thereafter, by cooling and solidifying the liquid embedding agent, both the biological tissue fragment22and the standard substance21are embedded in the same embedding agent23as illustrated inFIG.4Ain which parts that correspond to those ofFIG.3are given the same reference numerals, and the embedded block24integrated with the embedded block-preparing cassette1can be prepared (solidifying step). Finally, as illustrated inFIG.4Bin which the parts that correspond to those ofFIG.4Aare given the same reference numerals, the plate frame portion13provided on the lower surface3fof the frame portion3is removed, and the embedded block24is exposed to the outside of the embedded block-preparing cassette1. In this manner, by removing the plate frame portion13disposed in a quadrilateral shape in the embedded block-preparing cassette1, a rectangular parallelepiped embedded block24having four corners with a shape close to a right angle can be prepared. In addition, since the liquid embedding agent is solidified including that in the standard substance-positioning portion5in the opening4of the embedded block-preparing cassette1, the embedding agent23is integrated with the standard substance-positioning portion5, and thus it is possible to prevent the embedded block24from falling off from the embedded block-preparing cassette1.

(4) Tissue Section Selection Method of Present Invention

Next, an example of the tissue section selection method according to the present invention will be described. Here, similar to the “(1) Outline of Embedded Block-preparing Cassette of Present Invention”, a case where the state of the biological tissue is inspected by the immunohistochemical staining method will be described as an example. First, as illustrated inFIG.4B, an embedded block24in which both the biological tissue fragment22and the standard substance21are embedded in the same embedding agent23is prepared using the embedded block-preparing cassette1.

Next, the embedded block-preparing cassette1integrated with the embedded block24is set on a microtome. In the microtome, for example, the position of a cutting blade is set to a desired slicing thickness (for example, 4 μm), the embedded block24is sliced by the microtome, and a sheet-like embedded tissue section in which the cutting surfaces of the biological tissue fragment22and the standard substance21appear on the both surfaces is prepared. With the microtome, for example, by a cutting blade that moves up and down in one stroke, the embedded tissue section is excised from the embedded block24per each stroke.

Next, as illustrated inFIG.5, by stretching and mounting the sheet-like embedded tissue section44on a substrate46, such as a slide glass, a tissue section-mounting board40is prepared. The embedded tissue section44is dried on the substrate46. Here, the embedded tissue section44is formed in a rectangular shape having four corners of approximately a right angle. Therefore, by using the corners of the embedded tissue section44as a guide, an operator can grasp the position of the tissue section42in the embedded block24before cutting.

In the embedded tissue section44, the tissue section42, a thickness standard substance section41eas a standard substance section, and marker standard substance sections41d,41c,41band41aas the same standard substance sections are exposed to the surface, and each circumferential side of the tissue section42, the thickness standard substance section41e, and the marker standard substance sections41d,41c,41band41ais configured to be surrounded by an embedding agent section43.

Sequentially performed are, for example, an embedding agent-removing treatment for removing the embedding agent from the embedded tissue section44, a protease treatment with an antigen retrieval solution, an endogenous peroxidase treatment with a blocking reagent, and a staining treatment with the immunohistochemical staining method. In the staining treatment, for example, after causing an antigen-antibody reaction in the tissue section42using a primary antibody that binds to the HER2/neu protein in the tissue section42, a secondary antibody labeled polymer is bound to the primary antibody, and further the HER2/neu protein contained in the tissue section42is stained using a chromogenic reagent or the like, whereby the HER2/neu protein is specifically detected.

Next, the tissue section-mounting board40is placed on a stage of a microscope provided with a digital camera, the surface of the thickness standard substance section41eis photographed by focusing on the surface of the thickness standard substance section41e, and the image of the thickness standard substance section41eis obtained. At this time, the image is photographed at a predetermined magnification (in the present embodiment, the objective lens magnification ratio is 2.5). In addition, the image is generated based on the reflected light from the surface of the thickness standard substance section41e. The color tone of the image is also determined based on the reflected light.

Based on the image of the thickness standard substance section41e, the color tone of the thickness standard substance section41eis scored. In the present embodiment, by using a measurement program, pixels of a predetermined color tone (in the present embodiment, a color tone of a hue range of 272° to 305°, a chroma range of 0 to 51, a brightness range of a R value of 0 to 71, a brightness range of a G value of 0 to 77, and a brightness range of a B value of 0 to 77) are extracted from the image of the thickness standard substance section41e, and the color tone is scored by using the following Equation (1). Here, since the urethane foam stained in blue is used as the thickness standard substance21a, the above-described color tone range is selected.

Specifically, the extracted pixels are sorted by a chroma value, and the number of pixels that belong to the chroma range and an average brightness are calculated for each predetermined chroma range. The average brightness is calculated by averaging the average values of the R value, the G value and the B value of each pixel over all of the pixels that belong to each chroma range. The value obtained by subtracting the average brightness from an upper limit value 255 of the brightness value is divided by 255 to normalize the average brightness in each chroma range. A score is calculated by multiplying the average brightness normalized for each chroma range by a pixel ratio that is derived by dividing the number of pixels that belong to the chroma range by the number of extracted pixels, and then adding all the multiplication results.

Next, using a calibration curve that expresses a relationship between the score and the thickness of the thickness standard substance section41eprepared in advance, a thickness that corresponds to the score calculated by the above-described equation is calculated, and the thickness is used as the thickness of the tissue section42. In addition, when the thickness of the tissue section42is within a predetermined range (4 μm±0.4 μm in the present embodiment), the tissue section42on the tissue section-mounting board40is selected as a target to be inspected. Meanwhile, when the thickness of the tissue section42is not within the predetermined range, it is determined that the tissue section42on the tissue section-mounting board40is not to be inspected. In this manner, in the present embodiment, the tissue section42to be inspected is selected based on the color tone as a light signal from the thickness standard substance section41e.

Subsequently, the surface of the marker standard substance section41ais photographed while focusing on the surface of the marker standard substance section41ato obtain an image of the marker standard substance section41a. At this time, the image is photographed at a predetermined magnification (in the present embodiment, the objective lens magnification ratio is 40 times).

Based on the image of the marker standard substance section41a, the staining intensity of the marker standard substance section41ais scored based on the color tone of the marker standard substance section41a. In the present embodiment, by using the measurement program, pixels of a predetermined color tone (a color tone of a hue of 50° to 180°, a chroma range of 2 to 40, an R value of 10 to 180, a G value of 10 to 145, and a B value of 10 to 140) are extracted from the image of the marker standard substance section41a, the color tone is scored similarly to the thickness standard substance section41eusing the above-described Equation (1), and the score is regarded as the staining intensity score. In the present embodiment, the above-described color tone range is selected because the HER2/neu protein is stained brown.

Next, a graph is prepared, in which a vertical axis indicates the score of the staining intensity of the marker standard substance section41aand a horizontal axis indicates a thickness of the tissue section42, and which illustrates a calibration curve that represents a relationship between a staining intensity score of the marker standard substance section41aand a thickness of the tissue section42. Plotting is based on the thickness of the tissue section42calculated above and the staining intensity score of the marker standard substance section41a.

As a result of plotting, when the staining intensity score of the marker standard substance section41ais within a predetermined range (in the present embodiment, the range of the staining intensity score of the calibration curve of ±10%), staining of the HER2/neu protein in the tissue section42is appropriately performed, and the detection result of the marker is regarded as suitable. Accordingly, it is determined that the tissue section42can be inspected, and the tissue section42of the tissue section-mounting board40is selected as a target to be inspected.

Meanwhile, when the plot is not within the predetermined range, staining of the HER2/neu protein of the tissue section42is not appropriately performed, and there is a possibility that the detection result of the marker is not suitable, and, thus, there is a concern that an error may occur in inspection of the tissue section42, it is determined that the tissue section42of the tissue section-mounting board40is not to be inspected. In this manner, in the present embodiment, the tissue section42to be inspected is selected based on the color tone as a light signal from the marker standard substance section41a.

Accordingly, an operator who performs the inspection inspects the tissue section42by comparing the color tone of the selected tissue section42with the color tone of the marker standard substance sections41d,41c,41band41a.

Incidentally, instead of the marker standard substance section41a, the staining intensity score of any of the marker standard substance sections41d,41cand41bmay be calculated, and in a case where the staining intensity score is within a predetermined range, it may be determined that the inspection is possible. In a case where the marker standard substance section41dconsists of a negative cell line and the staining treatment is appropriately performed, the marker standard substance section cannot be stained. Thus, whether or not the staining is appropriately performed is determined based on whether or not the section41dis stained.

Each staining intensity score is calculated with respect to two or more marker standard substances selected among the marker standard substance sections41d,41c,41b, and41a, and when the staining intensity scores of the two or more marker standard substances are within a predetermined range, it may be determined that the inspection is possible. Furthermore, each staining intensity score is calculated with respect to the marker standard substance sections41d,41c,41band41a, and in a case where the staining intensity scores of all of the marker standard substance sections41d,41c,41band41aare within a predetermined range, it may be determined that the inspection is possible.

(5) Action and Effect

In the above-described configuration, the embedded block-preparing cassette1according to the embodiment of the present invention is configured such that the box body2is placed on the tray30into which the liquid embedding agent is poured when the embedded block24in which the biological tissue fragment22is embedded in an embedding agent23is prepared. The box body2of the embedded block-preparing cassette1includes: the frame3having the through hole6, which penetrates the upper surface3aand the lower surface3fin the region facing the biological tissue fragment22on the tray bottom34; and the standard substance-positioning portion5which is provided to face the tray bottom34and has the positioning hole5ainto which the standard substance21is inserted. In addition, in the standard substance-positioning portion5, the standard substance21is present within a height range from the lower end to the upper end of the biological tissue fragment22with respect to the tray bottom34when the standard substance21is inserted into the positioning hole5a.

Accordingly, when the embedded block24is prepared in the embedded block-preparing cassette1while maintaining the standard substance21to be present within the height range from the lower end to the upper end of the biological tissue fragment22by means of the standard substance-positioning portion5, the liquid embedding agent is poured into the opening4of the embedded block-preparing cassette1, whereby the embedded block24in which the biological tissue fragment22and the standard substance21are embedded in the same embedding agent23can be prepared. By using the embedded block-preparing cassette1, it is possible to prepare the embedded tissue section44in which the tissue section42, the thickness standard substance section41e, and the marker standard substance sections41d,41c,41band41aappear on the same surface by slicing the embedded block24. Accordingly, by using the embedded block-preparing cassette1, the tissue section to be inspected can be selected based on the color tone of the thickness standard substance section41eand, for example, the marker standard substance section41a, and the inspection can be performed only on the tissue section optimum for the inspection. Thus, it is possible to suppress occurrence of an error in the inspection result.

Furthermore, in the embedded block-preparing cassette1, the region in the opening4other than the region for forming the standard substance-positioning portion5is occupied by the through hole6, and when seen from above, the entire biological tissue fragment22is accommodated in the formation region of the through hole6. Accordingly, in the embedded block-preparing cassette1, when the embedded block24is prepared, the biological tissue fragment22can be pressed against the tray bottom34through the through hole6. Therefore, in the embedded block-preparing cassette1, it is possible to prevent the biological tissue fragment22from floating up, and it is possible to adjust the position of the tissue section42in accordance with the height range of the thickness standard substance21aand the marker standard substances21b,21c,21dand21e. Accordingly, by slicing the embedded block24prepared by using the embedded block-preparing cassette1, it is possible to reliably prepare the embedded tissue section44in which all of the tissue section42, the thickness standard substance section41e, and the marker standard substance sections41d,41c,41band41aappear on the same surface.

Furthermore, by using the embedded block-preparing cassette1, the rectangular parallelepiped embedded block24having four corners with a shape close to a right angle can be prepared by the plate frame portion13of the lower surface3fof the frame3. Accordingly, it is possible to set the outer circumferential shape of the plurality of embedded tissue sections44prepared by slicing the embedded block24to have a quadrilateral shape having four right-angled corners. When selecting the tissue section42to be inspected by attaching the embedded tissue section44to a slide glass, it is possible to more accurately grasp the position of the tissue section42to be confirmed even between different slides based on the four corners of the embedded tissue section44.

In the tissue section selection method of the present invention, the embedded block24is prepared by embedding all of the biological tissue fragment22, the thickness standard substance21a, and the marker standard substances21b,21c,21dand21ein the same embedding agent23(embedded block-preparing step), and the sheet-like embedded tissue section44in which all of the tissue section42, the thickness standard substance section41e, and the marker standard substance sections41d,41c,41band41aappear on the same surface is prepared by slicing the embedded block24(embedded tissue section preparing step). In addition, according to the tissue section selection method, the tissue section to be inspected is selected by calculating the thickness of the tissue section42based on the color tone (light signal) of the thickness standard substance section41e(tissue section selecting step, tissue section thickness determining step), and the tissue section42to be inspected is selected based on, for example, the color tone (light signal) of the marker standard substance section41aafter the tissue section thickness determining step (tissue section selecting step, marker suitability determining step).

Accordingly, the tissue section selection method enables calculation of the thickness of the tissue section42from the color tone of the thickness standard substance section41eby using the sheet-like embedded tissue section44in which the tissue section42and the thickness standard substance section41eappear on the same surface. Thus, according to the tissue section selection method, only the tissue section42having an optimum thickness for the inspection is selected, and the inspection can be performed only on the selected tissue section42, whereby, it is possible to suppress an error in the inspection result.

According to the tissue section selection method, it is possible to calculate a staining intensity score from the color tone of the marker standard substance section41aby using the embedded tissue section44in which the tissue section42and the marker standard substance section41aappear on the same surface. In this case, according to the tissue section selection method, the tissue section42to be inspected is selected based on the staining intensity score of the marker standard substance section41a. Therefore, according to the tissue section selection method, it is possible to more accurately determine the suitability of the detection result of the marker, and thus the inspection can be performed only on the tissue section42optimum for the inspection, thereby enabling suppression of an error in the inspection result.

Particularly, in the tissue section selection method, after selecting the tissue section42to be inspected by calculating the thickness of the tissue section42based on the color tone of the thickness standard substance section41e, a staining intensity score is further calculated based on the color tone of the marker standard substance section41a, and then the tissue section42to be inspected is selected. Thus, according to the tissue section selection method, it is possible to select only the tissue section42having a desired thickness and determine suitability of the detection result of the marker with respect to the selected tissue section42. Thus, it is possible to suppress occurrence of an error in the inspection result.

In the tissue section selection method, it is possible to prepare the embedded tissue section44in which the tissue section42and the marker standard substance sections41d,41c,41band41aappear on the same surface by slicing the embedded block24. Thus, it is possible to compare during inspection the stained tissue section42with the marker standard substance sections41d,41c,41band41aall of which were prepared and stained under the same condition. Therefore, it is possible to prevent the influence due to the difference in preparation conditions and/or the staining conditions, and to more accurately perform the inspection with respect to the tissue section42.

(6) Modified Example

Incidentally, the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the gist of the present invention. For example, the shape, size and the like of the tray30are not particularly limited, and can be appropriately selected in accordance with the embedded block-preparing cassette1. For example, those which are conventionally sold under the name of an embedding dish or the like can be used as the tray30, or the tray may be prepared in accordance with the shape, size and the like of the embedded block-preparing cassette. Further, the number of positioning holes5amay be the same as the number of standard substances21, or may be equal to or greater than the number of standard substances21.

In the above-described embodiment, while a case where the plate frame portion13is provided on the lower surface3fof the frame3has been described, the present invention is not limited thereto, and the plate frame portion13may not be provided on the lower surface3fof the frame3.FIG.6in which the parts that correspond to those ofFIG.4Bare given the same reference numerals is a schematic view illustrating a configuration when the embedded block24is prepared by using the embedded block-preparing cassette1in which the plate frame portion13is not provided on the lower surface3fof the frame3. As illustrated inFIG.6, the embedding agent23in the embedded block24can have the same outer shape as that of the accommodating portion31of the tray30. Therefore, for example, although the plate frame portion13is not provided, the embedding agent23has the four rounded corners in the rectangular parallelepiped shape. However, the operation of removing the plate frame portion13as illustrated inFIGS.4A and4Bbecomes unnecessary, and thus it is possible to reduce the work burden.

In the above-described embodiment, while the thickness standard substance21aand the marker standard substances21b,21c,21dand21eare used, and thus the standard substance-positioning portion5provided with the five positioning holes5ahas been described, the present invention is not limited thereto, and any standard substance-positioning portion provided with at least one positioning hole, such as one positioning hole or two positioning holes, may be used. In particular, it is desirable to have at least two positioning holes such that each of the thickness standard substance21aand the marker standard substance21ecan be embedded one-by-one in the embedded block24.

In the above-described embodiment, the standard substance-positioning portion5in which the three positioning hole-forming portions5f,5hand5jarranged in series and the two positioning hole-forming portions5gand5iarranged in series are arranged in two rows. But the present invention is not limited thereto, and the positioning hole-forming portions may be disposed in various positions such as in one row or three rows in the standard substance positioning portion. For example,FIGS.7A and7Bare schematic views illustrating an upper surface configuration of the embedded block-preparing cassettes51and61according to another embodiment. As illustrated inFIG.7A, the opening4in a box body52may be provided with a standard substance positioning portion55in which a plurality of positioning hole-forming portions5f,5g,5h,5iand5jare arranged in one row. Further, as illustrated inFIG.7B, four positioning hole-forming portions5g,5h,5iand5jmay be arranged in one row in the opening4of a box body62, and also may be provided a standard substance-positioning portion65in which the remaining one positioning hole-forming portion5fis disposed in a position different from the row of the positioning hole-forming portions5g,5h,5iand5j.

The height of the standard substance21has desirably such length that the standard substance21can be supported by the positioning hole-forming portion in a state where one end of the standard substance21abuts the tray bottom34although the present invention is not limited thereto. For example, in the above-described embodiment, the standard substance21is disposed over the entire height range of the biological tissue fragment22using the standard substance21that is equal to or higher than the height range of the biological tissue fragment22, but the present invention is not limited thereto. For example, a standard substance may be disposed only in a part of the region of the height range of the biological tissue fragment22using a standard substance shorter than the height range of the biological tissue fragment22.

In the above-described embodiment, a case where the hole shape of each positioning hole5ain the standard substance-positioning portion5is columnar and the standard substance21has a columnar shape in conformity with the columnar hole shape has been described. However, the present invention is not limited thereto, and the hole shape of each positioning hole5ain the standard substance-positioning portion5may be formed to be various other types, such as a quadrangular columnar shape or a polygonal columnar shape, and in conformity therewith, the standard substance21may also be formed to various other shapes, such as a quadrangular columnar shape or a polygonal columnar shape. Further, as long as the standard substance21can be supported by being inserted into the positioning hole5a, the shape of the positioning hole-forming portion, the shape of the positioning hole, and the shape of the standard substance are not particularly limited, and for example, the shape of the positioning hole and that of the standard substance may be different.

In the above-described embodiment, while a case where both the standard substance-positioning portion5and the through hole6are provided in the region in the opening4has been described, the present invention is not limited thereto. For example, different from the opening4in which the through hole6is formed, an opening which penetrates the upper surface and the lower surface may be provided in the frame3, and the standard substance-positioning portion5may be provided in the opening.

In the above-described embodiment, while a case where the quadrilateral through hole6is provided in one of the regions in the opening4divided into two by the horizontal bar9has been described, the present invention is not limited thereto, and, for example, a through hole having a circular, triangular, elliptical or polygonal shape, or the like may be provided. For example,FIG.8Ais a schematic view illustrating the upper surface configuration of an embedded block-preparing cassette71according to another embodiment. As illustrated inFIG.8A, a plate-like through hole-forming portion77having a circular through hole76may be provided in the opening4of a box body72. In addition, in the above-described embodiment, although a case where one through hole6is provided has been described, the present invention is not limited thereto. For example,FIG.8Bis a schematic view illustrating the upper surface configuration of an embedded block-preparing cassette81according to another embodiment. As illustrated inFIG.8B, in a partial region of the opening4of a box body82, may be provided a cross-shaped horizontal bar87having a cross shape in which two prismatic members are orthogonal to each other, and a through hole86divided into four.

In the above-described embodiment, while a case where the positioning hole-forming portions5f,5g,5h,5i, and5jare fixed to the inner wall3bof the frame3by the horizontal bar9provided on the inner wall3bof the frame3or the connecting portion5mhas been described, the present invention is not limited thereto, and the positioning hole-forming portions5f,5g,5h,5iand5jmay be fixed to the inner wall3bof the frame3only by the horizontal bar9or only by the connecting portion5m. The positioning hole-forming portions5f,5g,5h,5iand5jmay be directly fixed to the inner wall3bof the frame3without using the horizontal bar9or the connecting portion5m.

In the above-described embodiment, the tissue section42to be inspected is selected based on the color tone of the thickness standard substance section41eafter performing the staining treatment by the immunohistochemical staining method. However, the present invention is not limited thereto, and after selecting the tissue section42to be inspected based on the color tone of the thickness standard substance section41e, a treatment by the immunohistochemical staining method may be performed only on the tissue section-mounting board40having the selected tissue section42.

In the above-described embodiment, the corner of the embedded tissue section44is used in order to more accurately grasp the observation position of the tissue section42between each slide. However, in order to manage the observation position with higher accuracy, may be used, for example, a microscope system having a position synchronization function of a slide glass having a reference for position management, a high accuracy XY stage having means for correcting a rotation error of the placed slide glass when the slide glass is placed, or the like.

In the above-described embodiment, the color tone of the thickness standard substance section41eand the staining intensity of the marker standard substance section41aare scored using the image taken by the digital camera and the above-described Equation (1). However, the present invention is not limited thereto, and the color tone of the thickness standard substance section41eand the color tone of the marker standard substance section41amay be visually observed using a microscope, and the score may be determined based on a color sample, a tissue section determination example or the like.

The above-described embodiment has been described by way of an example wherein the tissue section is selected to be used for the inspection method for specifically detecting the HER2/neu protein contained in the biological tissue by the immunohistochemical staining method and inspecting the state of the living body. However, the present invention is not limited thereto, and the present invention may be applied as a marker to a case of detecting ALK fusion protein, PD-L1, estrogen receptor, progesterone receptor, EGF receptor, somatostatin, S-100, and the like by an immune tissue staining method.

Furthermore, the present invention may be applied to a case of selecting a tissue section used for an inspection method for inspecting the state of a living body by specifically detecting specific genes (markers), such as HER2/neu, ALK, PD-L1, estrogen receptor and progesterone receptor contained in the tissue section by a fluorescence in situ hybridization (FISH) method or a chromogenic in situ hybridization (CISH) method.

In the FISH method, a marker in the tissue section is detected by binding the marker in the tissue section to a probe to which a fluorescent label is bound by hybridization, and then detecting fluorescence of the tissue section with a fluorescence microscope. In this case, a fluorescence intensity as a light signal from the standard substance section is scored, and the tissue section42to be inspected is selected based on the thickness of the tissue section42and the fluorescence intensity score.

In the CISH method, a marker in the tissue section is detected by binding the marker in the tissue section to a probe to which an alkaline phosphatase-labeled anti-digoxigenin (DIG) antibody or the like is bound by hybridization, and by detecting chromogenic development of the alkaline phosphatase substrate. In this case, a chromogenic intensity as a light signal from the standard substance section is scored, and the tissue section42to be inspected is selected based on the thickness of the tissue section42and the chromogenic intensity score.

In the case of the FISH method or the CISH method described above, the marker standard substance including the markers used for each method is prepared, and the marker standard substance may be inserted into the positioning hole5ain the embedded block-preparing cassette1during preparation of the embedded block.

In the above-described embodiment, a case where a pigment-containing urethane foam is used as the thickness standard substance21ahas been described. However, the present invention is not limited thereto, and various cells that express an endogenous protein can also be used as the thickness standard substance21a. In this case, the endogenous protein exhibiting a constant expression level in various cells is stained by the immunohistochemical staining, and the color tone of the staining intensity of the cells is detected as a light signal. As the cells, for example, cells that express endogenous proteins, such as β-actin, COX-4, GAPDH, lamin B1, PCNA, tubulin and CD34, can be used. More specifically, for example, HER2 positive cell line SK-BR-3, cell line MDA-MB-453, or Hela cells derived from human cervical cancer and the like can be used. Since the expression level of the endogenous protein is substantially constant in each cell, the amount of cells changes corresponding to the thickness of the thickness standard substance21a, and the expression level of the endogenous protein also changes. In addition, the color tone of the staining intensity by the immunohistochemical staining changes corresponding to the thickness of the cells. Therefore, by using the cell as the thickness standard substance21a, the thickness of the tissue section42can be calculated based on the color tone of the thickness standard substance21a, and the tissue section42used for the inspection can be selected.

In a case where the cell that expresses a marker such as, for example, HER2/neu protein in addition to the endogenous protein is used as the thickness standard substance21a, the thickness standard substance21acan also be used as a marker standard substance. In this case, first, the embedded tissue section44is prepared using the thickness standard substance21a, and the endogenous protein and the marker which are expressed in the thickness standard substance section41eare respectively stained by the immunohistochemical staining. Next, the surface of the thickness standard substance section41eis photographed. Subsequently, the staining intensity score of the endogenous protein and the staining intensity score of the marker are respectively calculated using the above-described Equation (1) from the image of the thickness standard substance section41e, and the ratio between the score of the endogenous protein and that of the marker is calculated. When the ratio of the scores is within a predetermined value range, it is determined that the staining treatment is appropriate, and the tissue section42is selected as a tissue section to be inspected. Since the endogenous protein and the marker are stained in different colors, the score of the endogenous protein and the score of the marker can be calculated from the image of one fragment of the thickness standard substance section41e.

In verification experiment 1, a blue pigment-containing urethane foam is used as the thickness standard substance, and a relationship between the color tone of the blue pigment-containing urethane foam and the thickness of the tissue section was verified. Incidentally, as illustrated inFIG.5, the embedded tissue section44has the embedding agent section43and the tissue section42formed on the same surface, and thus the thickness of the embedding agent section43was deemed as the thickness of the tissue section42. In addition, since it is considered that the embedded tissue section has no significant change in the gradient of the calibration curve to be prepared regardless whether or not the embedded tissue section includes a tissue section, a section in which the tissue section42is not embedded was used hereinafter as the embedded tissue section for verification.

(7-1-1) Preparation of Thickness Standard Substance

To a polyol which is a soft urethane foam material, a foaming agent, a foam stabilizer and a catalyst, a blue pigment (manufactured by Resino Color Industries) in an amount of 4.5% of the total mass was added and mixed by stirring. By foaming the mixed material after molding, a blue pigment-containing urethane foam having a size of 100 to 110 mm in length, 60 to 70 mm in width and 30 to 40 mm in height, and a weight of 35 to 45 g was prepared.

The blue pigment-containing urethane foam was cut in a height direction into a sheet with a width of approximately 5 mm such that a cutting surface is parallel to the lateral direction of the blue pigment-containing urethane foam. Subsequently, from the sheet-like blue pigment-containing urethane foam, a part of the vicinity of the center where the number of bubbles contained in the blue pigment-containing urethane foam is small was excised, and sheet-like small urethane foam fragments having a thickness of approximately 5 mm were prepared.

The small urethane foam fragments were placed in a commercially available paraffin-embedded block-preparing cassette EB-W (manufactured by Olympus), and an immobilization treatment is performed using a closed type automatic immobilization embedding apparatus Tissue-Tek VIP (manufactured by Sakura Finetek Japan). A thickness standard substance was prepared by hollowing out the immobilized small urethane foam fragment into a columnar shape by using tissue micro-arrayer apparatus KIN-2-type (manufactured by Azumaya Medical Instruments Co., Ltd.).

(7-1-2) Preparation of Embedded Block Containing Thickness Standard Substance

In order to prepare an embedded tissue section for verification in which the tissue section42is not embedded, the embedded block-preparing cassette1was placed on the tray30without placing a biological tissue section on the tray bottom34. The thickness standard substance21awas inserted into the positioning hole5ain the embedded block-preparing cassette1, and then liquefied paraffin was poured from the opening4of the embedded block-preparing cassette1. After the paraffin was cooled and solidified, the embedded block-preparing cassette1was removed from the tray30, and the plate frame portion13was removed to obtain an embedded block in which only the thickness standard substance21awas embedded.

(7-1-3) Preparation of Embedded Tissue Section for Verification

The embedded block in which only the thickness standard substance21awas embedded was set in the microtome (manufactured by Daiwa Koki Kogyo Co., Ltd.). The slicing thickness of the microtome was set to 2 μm, the embedded block was sliced by the microtome, and ten embedded tissue sections for verification having a nominal thickness (hereinafter, referred to as nominal thickness) of 2 μm were prepared. The thickness standard substance sections41ewere exposed on both surfaces of the embedded tissue sections for verification.

Similarly, ten embedded tissue sections for verification each of which has a nominal thickness of 3 μm, 4 μm, 5 μm, and 6 μm were prepared. The embedded tissue sections for verification were attached to a slide glass (manufactured by Muto Glass Industrial Co., Ltd., Japan) one by one, and then dried at 37° C. overnight. In addition, the embedded tissue sections for verification have the same configuration as the embedded tissue section illustrated inFIG.5except that they do not include the tissue section and the marker standard substance section, and the circumference of the thickness standard substance section41eis surrounded by a sheet-like embedding agent section.

(7-1-4) Preparation of Calibration Curve for Calculating Thickness of Tissue Section

A slide glass with the embedded tissue section for verification was placed on a sample stage of the microscope system (manufactured by Nikon Co., Ltd.) maintained horizontally, and the height of the embedding agent section from the board was measured at four locations using optical interferometry by means of Vert Scan (registered trademark, manufactured by Ryoka System Co., Ltd., Ver. 1.0.3). The average value of the height measured at four locations was regarded as the thickness of the tissue section. The measurement conditions of Vert Scan were set as follows: the wavelength filter: 530White, the two-beam interference objective lens magnification: 10 times, the measurement mode: Wave mode.

A method for measuring the thickness of the embedding agent section will be specifically described. The XY stage, Z stage, and tilt stage of the sample stage of the microscope system were adjusted such that one of the four sides of the embedding agent section was projected on a measurement screen of a monitor attached to the microscope system, and the sample stage of the microscope was moved. A vertically moving handle was operated and adjusted to obtain the strongest contrast in the interference fringes in the image displayed on the monitor. The gradient of the sample stage was adjusted so that the number of the interference fringes in the image of the monitor becomes approximately 2 to 7. Measurement was performed, and whether or not the slide glass was placed horizontally was further confirmed by using as an indication that the color tone of the slide glass region in the measurement screen of the monitor is constant.

Subsequently, a partial region of the slide glass surface used as a reference for the thickness of the embedding agent section was designated, and a correction treatment for removing the influence of surface roughness and waviness of the designated region was performed. A partial region of the surface of the embedding agent section was designated, and the height of one side of the embedding agent section was measured with respect to the designated region of the slide glass surface after the correction treatment. The operation was also performed on the other three sides of the embedding agent section, and the average value of the measured heights of the four sides was regarded as the thickness of the tissue section.

Similarly, the thickness of the tissue section was calculated for all of the prepared embedded tissue sections for verification. Excluded were those embedded tissue sections for verification in which a difference in thickness of any one of the four sides of the embedding agent section from the nominal thickness exceeds 2.0 μm, and those embedded tissue sections for verification in which the thickness of the tissue section is calculated to have an existence probability of less than 0.3% when the variation in thickness is regarded as a normal distribution. As a result, a total of forty-two embedded tissue sections for verification were obtained, which include seven embedded tissue sections for verification having a nominal thickness of 2 μm, seven embedded tissue sections for verification having a nominal thickness of 3 μm, nine embedded tissue sections for verification having a nominal thickness of 4 μm, ten embedded tissue sections for verification having a nominal thickness of 5 μm, and nine embedded tissue sections for verification having a nominal thickness of 6 μm.

With respect to the forty-two embedded tissue sections for verification, by performing a deparaffinization treatment by a general method that has been already established, only the thickness standard substance section41ewas allowed to be present on the slide glass by removing the embedding agent section. In order to prevent the thickness standard substance section41efrom drying over time, a mounting medium was dropped onto the slide glass to cover the thickness standard substance section41ewith a cover glass.

Next, a slide glass on which the thickness standard substance section41ewas mounted was placed on the stage of the microscope (manufactured by Carl Zeiss Co., Ltd.) provided with a digital camera (manufactured by Canon Inc., EOS-1D), and the surface of the thickness standard substance section41ewas photographed by focusing on the surface of the thickness standard substance section41e. Thus, an image of the thickness standard substance section41ewas obtained (photographing conditions: objective lens magnification: 2.5 times, shutter speed: 1/250, ISO: 800, LED: 19.9%, WB correction: A9/G9).

Based on the image of the thickness standard substance section41e, the color tone of the thickness standard substance section41ewas scored using a measurement program (manufactured by Canon Inc.). Specifically, by using the measurement program, the pixels of the color tone in a hue range of 272° to 305°, a chroma range of 0 to 51, a brightness range of a R value of 0 to 71, a brightness range of a G value of 0 to 77, and a brightness range of a B value of 0 to 77 were extracted from the image of the thickness standard substance section41e, and the color tone was scored by using the above-described Equation (1).

In addition, as illustrated inFIG.9, the result was plotted, wherein a horizontal axis indicates a thickness of the tissue section (height average value of the embedding agent section), and a vertical axis indicates a score of the color tone of the thickness standard substance section41e. A linear approximate equation was calculated by the least square method based on the plot, and was regarded as a calibration curve. By the linear approximate equation calculated by the least square method, it was possible to prepare a calibration curve having a high correlation coefficient.

In this manner, since the thickness of the tissue section and the score of the color tone of the thickness standard substance section41eare in a proportional relationship, it is possible to calculate from the color tone of the thickness standard substance section41e, by using the calibration curve, the thickness of the tissue section that is embedded in the same embedded tissue section as the thickness standard substance section41e.

In addition, for example, with respect to the embedded tissue section prepared by setting the slicing thickness by the microtome to 4 μm, it is possible to calculate the thickness of the tissue section from the color tone of the blue pigment-containing urethane foam (thickness standard substance section41e) on the slide glass after attaching the embedded tissue section to the slide glass and effecting a deparaffinization treatment and a staining treatment. The tissue section in which the thickness is within a predetermined thickness range centering on 4 μm can be selected as a tissue section to be inspected. In this manner, it was possible to confirm that the calibration curve that represents a relationship between the thickness of the tissue section and the color tone of the thickness standard substance could be an index for selecting the tissue section to be inspected.

In verification experiment 2, by using cells that express β-actin as the thickness standard substance21a, the relationship between the color tone of the cells and the thickness of the tissue section42was verified. In addition, since the embedded tissue section44has the embedding agent section43and the tissue section42formed on the same surface, as illustrated inFIG.5, the thickness of the embedding agent section43was regarded as the thickness of the tissue section42. Further, since it is considered that the embedded tissue section44has no significant change in the gradient of the calibration curve to be prepared between the cases where an embedded tissue section44includes the tissue section42and does not include the tissue section42, an embedded tissue section in which the tissue section42is not embedded was used hereinafter as the embedded tissue section for verification.

(7-2-1) Preparation of Anti-β-Actin Rabbit Polyclonal Antibody

Antiserum was collected from a rabbit immunized with β-actin synthetic peptide, and anti-β-actin rabbit polyclonal antibody was prepared by antibody purification. A marker of a control sample was stained by immunohistochemical staining by using this antibody as a primary antibody and the staining results were evaluated. it was confirmed that it was possible to stain the marker with a smaller amount of this antibody compared with the case where an existing anti-β-actin monoclonal antibody (c4 SC-47778, manufactured by Santa Cruz Biotechnology, Inc.) was used. In the verification experiment 2, the anti-β-actin rabbit polyclonal antibody prepared as described above was used.

(7-2-2) Preparation of Thickness Standard Substance

In the verification experiment 2, an HER2 positive cell line SK-BR-3 (corresponding to HER2 IHC score 3+) was used as a cell. First, a paraffin-embedded block in which only the SK-BR-3 cell line was embedded in paraffin was prepared, and the paraffin-embedded block was hollowed out using a tissue arrayer apparatus type KIN-2 (manufactured by Azumaya Medical Instruments Co., Ltd.) to give a thickness standard substance21a.

(7-2-3) Preparation of Embedded Block Containing Thickness Standard Substance

In order to prepare an embedded tissue section for verification in which the tissue section42is not embedded, here, the embedded block-preparing cassette1was placed on the tray30without placing the biological tissue fragment22on the tray bottom34. The thickness standard substance21awas inserted into the positioning hole5ain the embedded block-preparing cassette1, and then liquefied paraffin was poured from the opening4of the embedded block-preparing cassette1. After the paraffin was cooled and solidified, the embedded block-preparing cassette1was removed from the tray30, and the plate frame portion13was removed to obtain an embedded block in which only the thickness standard substance21awas embedded.

(7-2-4) Preparation of Embedded Tissue Section for Verification

An embedded block in which only the thickness standard substance21awas embedded was set in the microtome (manufactured by Daiwa Koki Kogyo Co., Ltd.). The slicing thickness of the microtome was set to 2 μm, and the embedded block was sliced by the microtome to give ten embedded tissue sections for verification having a nominal thickness (hereinafter, referred to as nominal thickness) of 2 μm. The thickness standard substance sections41ewere exposed on both surfaces of the embedded tissue sections for verification.

Similarly, ten embedded tissue sections for verification were prepared for each nominal thickness of 3 μm, 4 μm, 5 μm, and 6 μm, and fifty (50) in total of embedded tissue sections for verification were prepared. The embedded tissue sections for verification were attached to the slide glass (manufactured by Muto Glass Industrial Co., Ltd.) one by one, and then dried at 37° C. overnight. Incidentally, the embedded tissue section for verification has the same configuration as the embedded tissue section44illustrated inFIG.5except that the tissue section and the marker standard substance section are not included, and the circumference of the thickness standard substance section41eis surrounded by the sheet-like embedding agent section43.

(7-2-5) Preparation of Calibration Curve for Calculating Thickness of Tissue Section

In the same manner as that in the verification experiment 1, the height of the embedding agent section of the embedded tissue section for verification was measured, and the thickness of the embedding agent section was calculated. After calculating the thickness of the embedding agent section for each embedded tissue section for verification, the deparaffinization treatment and antigen retrieval treatment (100° C., 40 minutes, pH 6) were performed according to a general method already established on the embedded tissue section for verification attached to the slide glass. After removing the endogenous peroxidase, the thickness standard substance section41ewas washed with PBS, and a primary antibody reaction with the anti-actin rabbit polyclonal antibody prepared in the “(7-2-1) Preparation of Anti-β-Actin Rabbit Polyclonal Antibody” was performed on β-actin expressed in HER2 positive cells in the thickness standard substance section41e. The thickness standard substance section41ewas washed again, and Simple Stain MAX-PO (MULTI) (manufactured by Nichirei Biosciences Inc.) was dropped onto the thickness standard substance section41eat a concentration of 4 μg/ml, and secondary antibody reaction was performed at room temperature for 30 minutes. The thickness standard substance section41ewashed again was subjected to DAB staining by a general method and washed again, and nuclear staining was performed using Mayer's Hematoxylin solution. After the thickness standard substance section41ewas washed and subjected to dehydration, permiation, and medium-mounting, it was covered with a cover glass.

The thickness standard substance section41eon which the above-described β-actin staining treatment was performed was placed on the slide glass and the slide glass was placed on the sample stage of the microscope (manufactured by Carl Zeiss), and by focusing on the cell membrane of the cell in which the stain of DAB was confirmed by the objective lens at a magnification ratio of 40, the thickness standard substance section41ewas photographed by the camera (manufactured by Canon Inc., EOS-1D) mounted on the microscope (shutter speed: 1/250, ISO: 800, LED: 19.9%, WB correction: A9/G9). From the photographed image, pixels having a specific color tone (hue: 272-305°, chroma range: 0 to 51, R: 0 to 71, G: 0 to 77, B: 0 to 77) were extracted by using the measurement program (manufactured by Canon Inc.), and the color tone of the staining intensity of the thickness standard substance section41ewas scored using the above-described Equation (1).

Then, as illustrated inFIG.10, the result was plotted, wherein a horizontal axis indicates the thickness of the tissue section (height average value of the embedding agent section), and a vertical axis indicates the score of the color tone of the thickness standard substance section41e, and a linear approximate equation was calculated by the least squares method based on the plot, and this was regarded as the calibration curve. In the linear approximate equation calculated by the least squares method, it was possible to prepare a calibration curve having a high correlation coefficient.

Accordingly, since the thickness of the tissue section and the score of the color tone of the staining intensity of the thickness standard substance section41ewere in a proportional relationship, it is possible to calculate the thickness of the tissue section embedded in the same embedded tissue section as the thickness standard substance section41eby using the calibration curve from the color tone of the thickness standard substance section41e.

In addition, for example, with respect to the embedded tissue section prepared by setting the slicing thickness by the microtome to 4 μm, it is possible, by attaching the embedded tissue section to the slide glass, to calculate the thickness of the tissue section from the color tone of the stained cell (thickness standard substance section41e) which express β-actin on the slide glass after a deparaffinization treatment and a staining treatment. The tissue section in which the thickness is within a predetermined thickness range centering on 4 μm can be selected as a tissue section to be inspected. Thus, even in a case where the cell which expresses endogenous protein was used as the thickness standard substance, it was confirmed that the calibration curve that represents a relationship between the thickness of the tissue section and the color tone of the thickness standard substance could be an index for selecting the tissue section to be inspected.

In verification experiment 3, a relationship between the staining intensity score based on the color tone of the stained marker standard substance and the thickness of the tissue section was verified. The thickness of the tissue section is calculated using the same method as that in the verification experiment 1. In addition, following the verification experiment 1, an embedded tissue section for verification that did not contain a tissue section and a thickness standard substance was used as the embedded tissue section.

(7-3-1) Preparation of Marker Standard Substance

In the verification experiment 3, similar to the above-described embodiment, a marker standard substance21bmade from the cell line MDA-MB-231 that exhibits negative staining result, a marker standard substance21cmade from the cell line MDA-MB-175VII that exhibits a staining result of 1+, a marker standard substance21dmade from the cell line MDA-MB-453 that exhibits a staining result of 2+, and a marker standard substance21emade from the cell line SK-BR-3 that exhibits a staining result of 3+, were provided as a marker standard substance that serves as a reference for the suitability of the marker detection results.

Specifically, control cell embedded blocks, in which each of the above-described cell lines was embedded in the embedding agent consisting of paraffin, were respectively prepared, and each of the control cell embedded blocks was hollowed out into a columnar shape using BIOPSY PUNCH (manufactured by Kai Industries Co., Ltd.) to obtain four types of marker standard substances.

(7-3-2) Preparation of Embedded Block Containing Marker Standard Substance

In order to prepare an embedded tissue section for verification in which the tissue section42is not embedded, here, the embedded block-preparing cassette1was placed on the tray30without placing the biological tissue fragment on the tray bottom34. Each of the marker standard substances21b,21c,21d, and21ewas inserted into each of the positioning holes5ain the embedded block-preparing cassette1, and then liquefied paraffin was poured thereto from the opening4of the embedded block-preparing cassette1. After the paraffin was cooled and solidified, the embedded block-preparing cassette1was removed from the tray30, and an embedded block in which the four types of marker standard substances21b,21c,21d, and21ewere embedded in the same embedding agent was obtained.

(7-3-3) Preparation of Embedded Tissue Section for Verification

The embedded block in which only the four types of marker standard substances21b,21c,21d, and21ewere embedded was set in the microtome (manufactured by Yamato Kohki Industrial Co., Ltd.), the embedded block was sliced while the slicing thickness was 2 μm, 3 μm, 4 μm, and 5 μm, and five embedded tissue sections for verification were prepared for each thickness. The marker standard substance sections41d,41c,41b, and41awere exposed on both surfaces of the embedded tissue sections for verification. In addition, the embedded tissue sections for verification were attached to one slide glass (manufactured by Matsunami Glass Industrial Co., Ltd.) one by one.

(7-3-4) Preparation of Calibration Curve for Evaluating Whether or not Detection is Suitable

In the same manner as in the verification experiment 1, the height of the embedding agent section of the embedded tissue section was measured. After calculating the thickness of the embedding agent section for each embedded tissue section, a deparaffinization treatment, an antigen retrieval treatment, and an immunohistochemical staining treatment were performed by using Histofine HER2 Kit (POLY) (manufactured by Nichirei Biosciences Inc.). In order to prevent the marker standard substance section from drying over time, a mounting medium was dropped onto the slide glass to cover the marker standard substance sections41d,41c,41b, and41awith a cover glass.

By using a microscope (manufactured by Carl Zeiss, Axio Imager.Z2) provided with a digital camera (manufactured by Canon Inc., EOS-1D), three pieces of still image of each of the slides of the marker standard substance sections41d,41c,41b, and41awere respectively photographed (photographing conditions: objective lens magnification: 40 times, aperture: 0.90, shutter speed: 1/125, ISO: 800, LED: 19.9%, WB correction: A9/G9).

Based on the photographed still images, the staining intensities of the four types of marker standard substance sections41d,41c,41b, and41awere respectively scored based on the color tone of the marker standard substance sections41d,41c,41b, and41a. Specifically, by using a measurement program (manufactured by Canon Inc.), pixels of a predetermined color tone (a color tone having a hue of 50° to 180°, a chroma range of 2 to 40, an R value of 10 to 180, a G value of 10 to 145, and a B value of 10 to 140) were extracted from the still images of the marker standard substance sections41d,41c,41b, and41a, and the color tone was scored using the above-described Formula (1). Scoring was performed on all of the marker standard substance sections41d,41c,41b, and41a.

As illustrated inFIG.11, the results were plotted, wherein a horizontal axis indicates the thickness of the tissue section (average height value of the embedding agent section) and a vertical axis indicates the staining intensity score. In addition, for each of the marker standard substance sections41d,41c,41b, and41a, a linear approximate equation was calculated by the least squares method based on the plot, and this was regarded as a calibration curve. Incidentally, the negative marker standard substance section41dwas not stained and thus is not illustrated inFIG.11. In the drawing, Score 1 corresponds to the marker standard substance section41c, Score 2 corresponds to the marker standard substance section41b, and Score 3 corresponds to the marker standard substance section41a. As illustrated inFIG.11, by the linear approximate equation calculated by the least squares method, it was possible to prepare a calibration curve having a high correlation coefficient.

Each calibration curve illustrated inFIG.11represents a staining intensity score based on the color tones of the marker standard substance sections41c,41b, and41awhen the tissue section is appropriately stained. Therefore, by plotting the thickness of the tissue section and the staining intensity score of the marker standard substance sections41c,41b, and41aon the graph illustrating the calibration curve, it is possible to see whether the difference between the plot and the calibration curve is within an appropriate range. Accordingly, it is possible to confirm whether or not the tissue section is appropriately stained by the calibration curve, and to select the tissue section to be inspected. In this manner, it was possible to confirm that the calibration curve that represents the relationship between the thickness of the tissue section and the staining intensity scores of the marker standard substance sections41c,41b, and41acould be an index for selecting the tissue section to be inspected.

(8) Immunohistochemical Staining Method Using Tissue Section Selection Method

(8-1) Series of Work Using Tissue Section Selection Method of Present Invention

Next, regarding the immunohistochemical staining method using the above-described tissue section selection method, a flow of a series of work will be briefly described with reference toFIG.12. As illustrated inFIG.12, in the immunohistochemical staining method, first, by using the embedded block-preparing cassette1illustrated inFIG.1, the embedded block24in which both the biological tissue fragment22and the standard substance21are embedded in the same embedding agent23is prepared as illustrated inFIG.4B.

Then, the embedded block-preparing cassette1integrated with the embedded block24is set on the microtome, and the embedded block24is sliced by the microtome set to a desired slicing thickness (for example, 4 μm) to obtain a sheet-like embedded tissue section44. Thereafter, as illustrated on a left side inFIG.12, a tissue section-mounting board40in which the embedded tissue section44is stretched and mounted on the board46, such as, for example, a slide glass, is prepared.

In the tissue section-mounting board40thus prepared, the tissue section42, the thickness standard substance section41eas the standard substance section, and the marker standard substance sections41d,41c,41band41aas the same standard substance sections are exposed on the surface of the embedding agent section43.

Next, for example, the above-described embedding agent removing treatment, the protease treatment and the endogenous peroxidase treatment are sequentially performed. Thereafter, as the staining treatment by the immunohistochemical staining method, for example, after causing an antigen antibody reaction in the tissue section42using a primary antibody bound to the HER2/neu protein in the tissue section42, a secondary antibody labeling polymer is bound to the primary antibody, and the HER2/neu protein contained in the tissue section42is stained further using a chromogenic reagent or the like to specifically detect the HER2/neu protein.

Next, the process proceeds to step S10illustrated inFIG.12, and based on the color tone of the thickness standard substance section41e, suitability of the thickness of the tissue section42is determined. For the determination of the suitability of the thickness of the tissue section in step S10, for example, an image obtained by photographing the surface of the thickness standard substance section41eis obtained with a microscope provided with a digital camera, pixels having a predetermined color tone are extracted from the image of the thickness standard substance section41eby using the measurement program, and then the color tone is scored using the above-described Formula (1).

In addition, from the calibration curve that represents a relationship between the score of the thickness standard substance section41eand the thickness, the thickness that corresponds to the score calculated by the above-described Formula (1) is specified, and this thickness is regarded as the thickness of the tissue section42. As a result, when the thickness of the tissue section42is within a predetermined range (for example, 3 to 5 μm), it is determined that the thickness of the tissue section42is a thickness appropriate for the inspection, and the process proceeds to the next step S11.

Meanwhile, when the thickness of the tissue section42is out of the predetermined range (a thickness that corresponds to a score of less than 3 μm (<3 μm), or a thickness that corresponds to a score of more than 5 μm (>5 μm)), it is determined that the thickness of the tissue section42is a thickness inappropriate for the inspection, and thus the tissue section42is not inspected. The above-described immunohistochemical staining is performed on the other tissue section-mounting board40, and then, the same treatment as those described above is performed. This process flow is repeated until the tissue section42having a predetermined thickness is obtained.

When it is determined that the tissue section42has a thickness appropriate for the inspection based on the color tone of the thickness standard substance section41e, it is determined in step S11, for example, whether or not staining of the HER2/neu protein in the tissue section42is appropriately performed, based on the color tone of the marker standard substance section41a.

For the determination as to whether or not staining is appropriately performed, for example, an image is obtained by photographing the surface of the marker standard substance section41awith a microscope provided with a digital camera, and by using the measurement program, pixels having a predetermined color tone are extracted from the image of the marker standard substance section41a, and the color tone is scored using the above-described Equation (1).

In addition, from the calibration curve that indicates a relationship between the staining intensity score of the marker standard substance section41aand the thickness of the tissue section42, and based on the thickness of the tissue section42calculated above and the staining intensity score of the marker standard substance section41a, evaluation is effected as to whether or not the staining intensity score of the marker standard substance section41ais within a predetermined range referring to the calibration curve.

As a result, when the staining intensity score is within a predetermined range with respect to the calibration curve, it is determined that the staining of the HER2/neu protein in the tissue section42is appropriate, and the process proceeds to the next step S13. On the other hand, when the staining intensity score deviates from the predetermined range with respect to the calibration curve, it is determined that the staining of the HER2/neu protein in the tissue section42is inappropriate, and inspection is not effected on the tissue section42. And the above-described immunohistochemical staining is performed on the other tissue section-mounting board40and then, a treatment similar to those described above is performed. In this manner, the process flow of the immunohistochemical staining, step S10and step S11is repeated until a tissue section42having a predetermined staining intensity is obtained.

In step S13(determination of human specimen), with respect to the tissue section42in which the staining of the HER2/new protein is determined to be appropriate, the operator compares the color tone of the tissue section42with the color tone of the marker standard substance sections41d,41c,41band41a, and the tissue section42is inspected.

Here, a verification experiment was performed, wherein a series of operation illustrated inFIG.12was actually performed continuously, and the thickness of the tissue section and the staining intensity score of the marker standard substance section were examined and compared with the calibration curve of “Score 3” illustrated inFIG.11. Here, similar to the verification experiment 1, the blue pigment-containing urethane foam was used as the thickness standard substance, and the marker standard substance21emade from the cell line SK-BR-3 showing a staining result of 3+ was used as a marker standard substance that serves as a reference for the suitability of the marker detection result.

The embedded block in which the thickness standard substance21aand the marker standard substance21ewere embedded was set in the microtome (manufactured by Yamato Kohki Industrial Co., Ltd.), and the embedded block was sliced while setting the slicing thickness to 4 μm to give five embedded tissue sections for verification. The thickness standard substance section41eand the marker standard substance section41awere exposed on both surfaces of the embedded tissue section for verification. In addition, the embedded tissue sections for verification were attached one by one to a slide glass (manufactured by Matsunami Glass Industrial Co., Ltd.).

Next, a deparaffinization treatment, an antigen retrieval treatment, and an immunohistochemical staining treatment were performed by using Histofine HER2 Kit (POLY) (manufactured by Nichirei Biosciences Inc.). In order to prevent the marker standard substance section41afrom drying over time, a mounting medium was dropped onto the slide glass to cover the marker standard substance section41awith a cover glass.

Next, the above-described slide glass was placed on the stage of the microscope (manufactured by Carl Zeiss) provided with a digital camera (manufactured by Canon Inc., EOS-1D), and the surface of the thickness standard substance section41ewas photographed by focusing on the surface of the thickness standard substance section41e. Accordingly, an image of the thickness standard substance section41ewas obtained (photographing conditions: objective lens magnification: 2.5 times, shutter speed: 1/250, ISO: 800, LED: 19.9%, WB correction: A9/G9).

Based on the image of the thickness standard substance section41e, the color tone of the thickness standard substance section41ewas scored using a measurement program (manufactured by Canon Inc.). Specifically, by using the measurement program, the pixels of the color tone in a hue range of 272° to 305°, a chroma range of 0 to 51, a brightness range of a R value of 0 to 71, a brightness range of a G value of 0 to 77, and a brightness range of a B value of 0 to 77 were extracted from the image of the thickness standard substance section41e, and the color tone was scored by using the above-described Formula (1). In addition, based on the obtained score, the height of the thickness standard substance section41ewas measured from the calibration curve illustrated inFIG.9.

In addition, using a microscope (manufactured by Carl Zeiss, Axio Imager.Z2) provided with a digital camera (manufactured by Canon Inc., EOS-1D), slides of the still images of the marker standard substance section41awere respectively photographed (photographing conditions: objective lens: 40 times, aperture: 0.90, shutter speed: 1/125, ISO: 800, LED: 19.9%, WB correction: A9/G9).

Based on the photographed still image, the staining intensity of the marker standard substance section41awas scored based on the color tone of the marker standard substance section41a. Specifically, by using the measurement program (manufactured by Canon Inc.), pixels of a predetermined color tone (a color tone in a hue range of 50° to 180°, a chroma range of 2 to 40, an R value of 10 to 180, a G value of 10 to 145, and a B value of 10 to 140) were extracted from the still image of the marker standard substance section41a, and the color tone was scored using the above-described Formula (1).

In addition, as illustrated inFIG.13, the measurement result of each embedded tissue fragment for verification was plotted, wherein a horizontal axis indicates the thickness of the thickness standard substance section41e, and a vertical axis indicates the staining intensity score, and the result was compared with the calibration curve of “Score 3” illustrated inFIG.11. Incidentally, the “linear (HS standard Score 3)” inFIG.13indicates a calibration curve of “Score 3” inFIG.11. As a result of the comparison, the difference between the plot and the calibration curve was within an appropriate range, and it was possible to confirm whether or not the tissue section was appropriately stained in any of the embedded tissue sections for verification by the calibration curve. Accordingly, it was confirmed that it was possible to select the tissue section to be inspected by the tissue section selection method of the present invention.

(9) Immunohistochemical Staining Method Using Tissue Section Selection Method for Determining Fixed State of Tissue Section

(9-1) Preparation of Tissue Section-Mounting Board

Here, different from the above-described embodiment, the tissue section selection method for determining the state of the fixed tissue section will be described below. In the embedded block-preparing step of preparing an embedded block, a biological (living) tissue fragment collected from the living body is usually immersed in a fixing solution such as a formalin aqueous solution for a predetermined period of time or more (immersing step). As a typical fixing solution, a 10% formalin aqueous solution (4% formaldehyde solution) obtained by diluting a formalin stock solution with distilled water or a buffer is known.

Here, an appropriate period of time for immersing the biological tissue fragment in the fixing solution depends on the fixing solution, the size of the biological tissue fragment or the like. For example, in a case of staining the HER2/neu protein, it is recommended to immerse the biological tissue fragment in the fixing solution for 6 to 72 hours by using the above-described 10% formalin aqueous solution (the period of time for immersing the biological tissue fragment in the fixing solution is referred to as a fixing time). By immersing the biological tissue fragment in the fixing solution for the recommended fixing time, it is possible to optimally fix the tissue form or the antigen activity of the biological tissue fragment by the fixing solution.

In this manner, after the biological tissue fragment is immersed in the fixing solution for a predetermined period of time, the biological tissue fragment is placed at a predetermined position on the tray (placing step). In addition, the embedded block-preparing cassette is placed on the tray, and a liquid embedding agent such as paraffin that is liquefied by heating is poured from the embedded block-preparing cassette onto the tray (liquid embedding agent introducing step).

The liquid embedding agent is poured until the biological tissue fragment on the tray is covered (liquid embedding agent introducing step), and by cooling and solidifying the liquid embedding agent, the biological tissue fragment is embedded in the embedding agent, and an embedded block integrated with the embedded block-preparing cassette can be prepared (solidifying step).

In addition, the embedded block-preparing cassette integrated with the embedded block is set in the microtome, the embedded block is sliced by the microtome set to a desired slicing thickness (for example, 4 μm), and the sheet-like embedded tissue section is obtained.

Thereafter, as illustrated in the left side ofFIG.14, for example, by stretching and mounting the embedded tissue section91on the substrate46such as a slide glass, a tissue section-mounting board90is prepared. Incidentally, the embedded tissue section91of the present embodiment has a configuration in which the tissue section42is exposed on the surface and the circumference of the tissue section42is surrounded by the embedding agent section43.

(9-2) Determination Method of Fixed State of Tissue Section

Here, when preparing the embedded block, a fixing treatment for fixing the biological tissue fragment obtained from the living body is performed to give an optimal state as to the tissue form and the antigen activity. However, if, for example, the period of time for immersing the biological tissue fragment in the fixing solution is not sufficient, the biological tissue fragment is unlikely to be optimally fixed. When the biological tissue fragment is not optimally fixed, the tissue form and the antigen activity of the biological tissue fragment are not maintained, and then when inspecting the tissue section prepared from the biological tissue fragment by the immunohistochemical staining therafter, the tissue sections are unlikely to be stained, and there is a possibility that an error occurs in the inspection results.

Here, in the present embodiment, it is determined with respect to the tissue section-mounting board90as to whether or not the state of the tissue section42fixed by the fixing solution is optimal. In this case, a staining treatment is performed on the embedded tissue section91by an immunohistochemical staining, and based on the light signal from the endogenous protein expressed in the cells in the tissue section42, it is determined whether or not the state of the tissue section42fixed by the fixing solution is optimal. Hereinafter, the determination method of the state of the fixed tissue section42will be described.

In the present embodiment, for example, different from the staining treatment by the immunohistochemical staining method for staining the HER2/neu protein of the tissue section42, another staining treatment by the immunohistochemical staining method is performed on the tissue section-mounting board90, and an endogenous protein expressed in the cells in the tissue section42is stained. Here, the color tone at the time of staining of the endogenous protein on which the staining treatment by the immunohistochemical staining method is performed changes corresponding to the state of the biological tissue fragment fixed by the fixing solution which was performed when the embedded block was prepared. Based on the color tone of the endogenous protein contained in the tissue section42, the operator determines whether or not the fixing treatment on the tissue section42has been appropriately performed.

Here, the endogenous protein contained in the tissue section and used in the determination method is desirably endogenous protein in which an expression level does not substantially change depending on the cell, or endogenous protein stably expressed at an evaluated position. For example, this type of endogenous protein is desirably CD34 detected in capillary endothelial cells.

Since capillary blood vessels are generally present in the biological tissue fragments that are inspected for the presence or absence of tumor cells, such as breast cancer, the CD34 detected in the capillary endothelial cells is also stably expressed. In addition, since it is known that angiogenesis is generally accelerated around tumors, it can be said that in a case where there are tumor cells in the biological tissue fragment, the CD34 detected in the capillary endothelial cells is stably expressed.

In a case where the CD34 is applied as the endogenous protein expressed in the cells in the tissue section, an anti-CD34 antibody can be applied as an anti-endogenous protein antibody that stains the endogenous protein. As the anti-CD34 antibody, for example, the anti-CD34 rabbit polyclonal antibody can be applied.

When more specifically the above-described treatment steps are described, in the present embodiment, after obtaining the tissue section-mounting board90, for example, the above-described embedding agent removing treatment, the protease treatment, and the endogenous peroxidase treatment are sequentially performed. Thereafter, as the staining treatment by the immunohistochemical staining method, for example, after causing an antigen-antibody reaction in the tissue section42using a primary antibody that binds to the HER2/neu protein in the tissue section42, a secondary antibody-labeled polymer is bound to the primary antibody, and the HER2/neu protein contained in the tissue section42is stained further using a chromogenic reagent or the like to specifically detect the HER2/neu protein.

In addition, at this time, as another staining treatment by the immunohistochemical staining method, further, after causing an antigen-antibody reaction in the tissue section42using a primary antibody that binds to the endogenous protein expressed in the cells in the tissue section42, the secondary antibody-labeled polymer is bound to the primary antibody, and the endogenous protein contained in the tissue section42is stained further using a chromogenic reagent or the like to specifically detect the endogenous protein.

In this case, by changing the color tone of the HER2/neu protein contained in the tissue section42when it is stained and the color tone of the endogenous protein contained in the tissue section42when it is stained, it is possible to distinguish staining state of the HER2/neu protein from that of the endogenous protein.

Further, in a case of staining the HER2/neu protein contained in the tissue section42, it is confirmed by the verification experiment that, for example, the CD34 can be used as the endogenous protein used for evaluating the fixing state of the tissue section42. It has been confirmed that it is possible to avoid overlapping of the color tone of the stained HER2/neu protein and the color tone of the stained CD34 in a case where the CD34 is applied in the determination of the fixing state. Therefore, based on the color tone of the CD34 contained in the tissue section42, the operator can determine whether or not the fixing treatment for the tissue section42has been appropriately performed.

Here, a flow of a series of operation will be briefly described with reference toFIG.14regarding the immunohistochemical staining method using the tissue section selection method in the present embodiment. As illustrated inFIG.14, in the immunohistochemical staining method herein, a staining treatment for staining the HER2/neu protein contained in the tissue section42and a staining treatment for staining the endogenous protein contained in the tissue section42are respectively performed with respect to the tissue section-mounting board90, and the process proceeds to step S21.

In step S21, the state of the tissue section42fixed is determined based on the staining intensity of the endogenous protein that is contained in the tissue section42and changed by the staining treatment. Incidentally, in the determination of the fixed state of the tissue section42, for example, an image obtained by photographing the surface of the tissue section42is obtained using the microscope provided with a digital camera, and by using a measurement program, pixels having a predetermined color tone are extracted from the image of the tissue section42, and the color tone is scored using the above-described Formula (1).

Here, as illustrated inFIG.15, a graph (which will be described later) that represents a relationship between the score obtained from the color tone of the endogenous protein and the fixing time is prepared in advance. From the graph, a fixing time that corresponds to the score calculated by the above-described Equation (1) is calculated, and this value can be estimated as the fixing time of the tissue section42. As a result, when the fixing time of the tissue section42is equal to or longer than a predetermined time (for example, 24 hours or longer), the fixing time at the time of the fixing treatment performed on the tissue section42is optimal, and it is determined that the tissue section42is in a fixing state appropriate for the inspection. Then the process proceeds to the next step S22.

Meanwhile, when the fixing time of the tissue section42obtained from the graph is shorter than the predetermined period of time (when the fixing time that corresponds to the score is shorter than, for example, 24 hours), the fixing time for the tissue section42is insufficient, and it is determined that the tissue section is in a fixing state inappropriate for the inspection. Then the determining treatment is terminated without inspecting the tissue section42.

In step S21, when the tissue section42to be an inspection target is selected based on the color tone of the endogenous protein contained in the tissue section42, the operator, in step S22(human specimen determination), compares the color tone of the HER2/neu protein in the tissue section42with the control slide prepared in advance, and inspects whether or not the HER2/neu protein is present in the tissue section42.

(9-3) Action and Effects

In the tissue section selection method according to the present embodiment with the above-described configuration, the biological tissue fragment is immersed in a fixing solution for a predetermined time, and then the biological tissue fragment is embedded in an embedding agent to prepare the embedded block (embedded block-preparing step). Further, the embedded block is sliced to prepare the sheet-like embedded tissue section91in which the tissue section42appears on the surface (embedded tissue section-preparing step).

In the tissue section selection method, when the marker is specifically detected and the tissue section42is selected as an inspection target, the staining treatment is performed on the embedded tissue section91to stain the endogenous protein expressed in the cells in the tissue section42. Thus, based on the light signal from the endogenous protein expressed in the cells in the tissue section42, the state of the tissue section42fixed with the fixing solution is determined (fixing state determining step).

In other words, in the tissue section selection method according to the present embodiment, the fixing time of the tissue section42is estimated from the color tone of the endogenous protein contained in the tissue section42. Accordingly, in the tissue section selection method, only the tissue section42having an optimum fixing state for the inspection can be selected, and thus inspection can be performed only on the selected tissue section42. Accordingly, it is possible to suppress occurrence of an error in the inspection result.

(9-4) Modification Example

Incidentally, the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the gist of the present invention. While above-described embodiment describes a case where both the staining treatment for staining the HER2/neu protein in the tissue section42and the staining treatment for staining the endogenous protein expressed in the cells in the tissue section42are performed by the immunohistochemical staining method, the present invention is not limited thereto. For example, only the staining treatment for staining the endogenous protein expressed in the cells in the tissue section42may be performed. Further, after the fixing state determining step of determining the fixing state of the tissue section42, for example, the staining treatment by the immunohistochemical staining method for staining the HER2/neu protein in the tissue section42may be performed.

Furthermore, for example, the fixing state determining step for determining the fixing state of the tissue section42according to the present embodiment may be incorporated in the above-described “(8) Immunohistochemical Staining Method Using Tissue Section Selection Method”.

In this case, the step can be realized by providing the fixing state determining step illustrated in step S21ofFIG.14after the embedded tissue section preparing step of preparing the sheet-like embedded tissue section, before step S10(tissue section thickness determining step) in the flowchart illustrated inFIG.12, or after step S10(tissue section thickness determining step), or after step S11(marker suitability determining step).

More specifically, for example, by providing the step S21ofFIG.14after performing the staining treatment for staining the HER2/neu protein in the tissue section42and the staining treatment for staining the endogenous protein expressed in the cells in the tissue section42by the immunohistochemical staining method, before step S10, or after step S10, or after step S11illustrated inFIG.12, the fixing state of the tissue section42can be determined.

Further, the tissue section selection method may be such a method in which the following steps are combined: (i) the tissue section thickness determining step for determining the thickness of the tissue section42based on the color tone of the thickness standard substance section41eas illustrated in step S10inFIG.12, and (ii) the fixing state determining step for determining the fixing state of the tissue section42fixed by a fixing solution based on the color tone of the endogenous protein in the tissue section changed by another staining treatment as illustrated in step S21inFIG.14.

In this case, by using a tissue section-mounting board on which the tissue section42and the thickness standard substance section41eas the standard substance section are exposed on the surface of the embedding agent section43, the above-described step S10and step S21are performed.

Further, the tissue section selection method may be such a method in which the following steps are combined: (i) the marker suitability determining step for determining whether or not staining of the HER2/neu protein in the tissue section42is appropriately performed based on the color tone of the marker standard substance section41a, as illustrated in step S11inFIG.12, and (ii) the fixing state determining step for determining the state of the tissue section42fixed by the fixing solution based on the color tone of the endogenous protein in the tissue section changed by another staining treatment, as illustrated in step S21inFIG.14.

In this case, the above-described step S11and step S21are performed by using the tissue section-mounting board on which the tissue section42and the marker standard substance sections41d,41c,41band41aas the standard substance section are exposed on the surface of the embedding agent section43.

(9-5-1) Examination of Concentration of Anti-CD34 Rabbit Polyclonal Antibody

In verification experiment 5, a porcine mammary gland tissue was prepared, and the porcine mammary gland tissue was set in the microtome (manufactured by Yamato Kohki Industrial Co., Ltd.). The slicing thickness of the microtome was set to 4 μm, and the porcine mammary gland tissue was sliced to prepare a plurality of tissue sections for verification. After the tissue section for verification was attached to the slide glass (manufactured by Muto Glass Industrial Co., Ltd.), staining was performed on CD34 of the tissue section for verification with an anti-CD34 rabbit polyclonal antibody (Abcam Co.) having different concentrations.

As the anti-CD34 rabbit polyclonal antibodies with different concentrations, anti-CD34 rabbit polyclonal antibodies were diluted with PBS to prepare an antibody with a concentration of 1/100, an antibody with a concentration of 1/200, an antibody with a concentration of 1/400, an antibody with a concentration of 1/800, and an antibody with a concentration of 1/1600. In addition, the staining treatment for staining the porcine mammary gland tissue was performed using the five types of antibodies having different concentrations.

Next, the tissue section for verification after the staining treatment was encapsulated in a slide glass, and the slide glass was placed on the stage of the microscope (manufactured by Carl Zeiss) provided with a digital camera (manufactured by Canon Inc., EOS-1D), and the surface of the tissue section for verification was photographed by focusing on the surface of the tissue section for verification. Accordingly, images93a,93b,93c,93d, and93eillustrated inFIG.16were respectively obtained (photographing conditions: shutter speed: 1/250, ISO: 800, WB correction A9/G9, LED: 19.9%, aperture: 0.50, objective lens magnification: 40 times).

The image93ais an image obtained by photographing the surface of the tissue section for verification which was stained using the antibody having a concentration of 1/100, the image93bis an image obtained by photographing the surface of the tissue section for verification which was stained using the antibody having a concentration of 1/200, the image93cis an image obtained by photographing the surface of the tissue section for verification which was stained using the antibody having a concentration of 1/400, the image93dis an image obtained by photographing the surface of the tissue section for verification which was stained using the antibody having a concentration of 1/800, and the image93eis an image obtained by photographing the surface of the tissue section for verification which was stained using the antibody having a concentration of 1/1600.

Further, as a negative control, the same porcine mammary gland tissue as described above was prepared as the tissue section for verification, and similarly, the tissue section for verification was photographed to acquire an image93f.

From the results illustrated inFIG.16, in the tissue sections for verification on which the staining was performed using the antibody having a concentration of 1/100 and the antibody having a concentration of 1/200, it was possible to confirm that the color tone was deepened as illustrated in the images93aand93b. As described above, in a case where the color tone is deepened, for example, in a case where the HER2/neu protein or the like is further stained, there is a concern that the staining may also be affected. Therefore, it is desirable to use an antibody with a concentration lower than 1/200.

Meanwhile, in the tissue section for verification on which the staining was performed using an antibody having a concentration of 1/1600, the color tone was lightened, as illustrated in the image93e, substantially similar to the image93fof the negative control, and it was confirmed that the staining was insufficient. From the above, it was confirmed that the tissue section for verification was stained by using the anti-CD34 rabbit polyclonal antibody having a concentration of more than 1/200 and less than 1/1600, more preferably the antibody having a concentration of 1/400 or more and 1/800 or less.

(9-5-2) Evaluation of Staining Using Anti-CD34 Rabbit Polyclonal Antibody Regarding Fixing Time When Porcine Mammary Gland Tissue was Immersed in Fixing Solution

Next, a verification experiment was performed on the case where a porcine mammary gland tissue section for verification was stained using the above-described antibody having a concentration of 1/400 in order to confirm whether or not the color tone changes depending on the difference in fixing time when the porcine mammary gland tissue is immersed in a fixing solution.

Here, a part of the porcine mammary gland tissue was excised to obtain a plurality of tissue blocks, and each of the tissue blocks immediately after excision was respectively immersed in a fixing solution. A 10% formalin aqueous solution was used as the fixing solution. In addition, the time for immersing the tissue block in the fixing solution (fixing time) was changed for each tissue block.

The fixing time of the tissue block was set to 3 hours, 24 hours, 48 hours, 72 hours, and 1 week. After each time has elapsed, each tissue block was sliced using the microtome (manufactured by Yamato Kohki Industrial Co., Ltd.) set at a slicing thickness of 4 μm, and the tissue sections for verification were prepared.

After the tissue section for verification was attached to the slide glass (manufactured by Muto Glass Industrial Co., Ltd.), staining was performed with respect to the CD34 of the tissue section for verification using the anti-CD34 rabbit polyclonal antibody (Abcam Co.) having a concentration of 1/400, respectively.

Next, the slide glass in which a staining-treated tissue section for verification was encapsulated was placed on the stage of the microscope (manufactured by Carl Zeiss) provided with a digital camera (manufactured by Canon Inc., EOS-1D), and the surface of the tissue section for verification was photographed by focusing on the surface of the tissue section for verification. Thus, images94a,94b,94c,94d, and94eillustrated inFIG.17were obtained.

The image94ais an image obtained by photographing the surface of the tissue section for verification with a fixing time of 3 hours, the image94bis an image obtained by photographing the surface of the tissue section for verification with a fixing time of 24 hours, the image94cis an image obtained by photographing the surface of the tissue section for verification with a fixing time of 48 hours, the image94dis an image obtained by photographing the surface of the tissue section for verification with a fixing time of 72 hours, and the image94eis an image obtained by photographing the surface of the tissue section for verification with a fixing time of 1 week.

From the image94a, it was confirmed that sufficient staining was not obtained with respect to the tissue section for verification with a fixing time of 3 hours. Meanwhile, from the image94b, it was confirmed that the tissue section for verification with a fixing time of 24 hours was stained. Further, from the images94b,94c,94d, and94e, it was confirmed that no significant difference was observed in staining among the cases where the fixing time was set in a range of from 24 hours to 1 week.

From the above, it was confirmed that there was a large difference in staining between the case where the fixing time was set to 3 hours and the case where the fixing time was set to 24 hours. Accordingly, it was confirmed that it is possible to determine the fixing time of the tissue section by the difference in color tone by staining the tissue section using the anti-CD34 rabbit polyclonal antibody having a concentration of 1/400.

(9-5-3) Preparation of Graph for Determining Suitability of Fixing State of Tissue Section

Next, preparation of a graph (FIG.15) for determining suitability of the fixing state of the tissue section will be described. Immediately after cutting out a plurality of tissue blocks from the porcine mammary gland tissue, each of the tissue blocks was immersed in the fixing solution. The fixing time of the tissue block was set to 3 hours, 24 hours, 48 hours, 72 hours, and 1 week. After each time has elapsed, each tissue block was sliced using the microtome (manufactured by Yamato Kohki Industrial Co., Ltd.) set at a slicing thickness of 4 μm, to give the tissue sections for verification.

The tissue sections for verification were attached to the slide glass (manufactured by Muto Glass Industrial Co., Ltd.). Two such slide glasses were respectively provided for each fixing time. The anti-CD34 rabbit polyclonal antibody (Abcam Co.) was diluted with PBS to prepare an antibody having a concentration of 1/400, and staining was performed on CD34 in each tissue section for verification using the antibody.

Next, the tissue section for verification after the staining treatment was encapsulated in a slide glass and the slide glass was placed on the stage of the microscope (manufactured by Carl Zeiss) provided with a digital camera (manufactured by Canon Inc., EOS-1D), and the surface of the tissue section for verification was photographed by focusing on the surface of the tissue section for verification. Images98a,98b,98c,98d, and98eillustrated inFIG.21, for example, were obtained with photographing conditions: shutter speed of 1/250, ISO of 800, WB correction of A9/G9, LED of 19.9%, aperture of 0.50, and objective lens of 40 times.

Incidentally, the image98ais an image obtained by photographing the surface of the tissue section for verification with a fixing time of 3 hours, the image98bis an image obtained by photographing the surface of the tissue section for verification with a fixing time of 24 hours, the image98cis an image obtained by photographing the surface of the tissue section for verification with a fixing time of 48 hours, the image98dis an image obtained by photographing the surface of the tissue section for verification with a fixing time of 72 hours, and the image98eis an image obtained by photographing the surface of the tissue section for verification with a fixing time of 1 week. The image98fis an image obtained by enlarging a part of the image98cobtained by photographing the surface of the tissue section for verification with a fixing time of 48 hours.

Based on the obtained images98a,98b,98c,98d, and98e, the stained portions stained to brown color were scored. Specifically, by using the measurement program (manufactured by Canon Inc.), for example, pixels of a predetermined color tone (thinning out of pixels in the image: 1/64, a chroma range: 12 to 25, a hue range: 90° to 120°, and RGB ranges: a R value of 85 to 130, a G value of 65 to 120, and a B value of 65 to 120) were extracted from each of the images98a,98b,98c,98dand98e, and the color tone was scored using the above-described Formula (1).

As illustrated inFIG.15, wherein a horizontal axis indicates a fixing time of the tissue section (specimen fixing time) and a vertical axis indicates a staining intensity score (brown score), the results are plotted to give a graph illustrated inFIG.15.

As a result, as illustrated inFIG.15, it was possible to express as a graph the staining intensity score based on the color tone of the tissue section when the fixing treatment was appropriately performed on the tissue section. Therefore, by plotting the staining intensity score obtained based on the tissue section in the graph, the fixing time can be estimated. Accordingly, it is possible to confirm whether or not the fixing treatment is appropriately performed on the tissue section by the graph, and to select the tissue section to be inspected. In this manner, it was possible to confirm that the graph that expresses a relationship between the staining intensity score of the tissue section and the fixing time could be an index for selecting the tissue section to be inspected.

REFERENCE SIGNS LIST