Patent Publication Number: US-6703247-B1

Title: Apparatus and methods for efficient processing of biological samples on slides

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a continuation-in-part of application U.S. Ser. No. 08/780,029 filed Dec. 23, 1996 now U.S. Pat. No. 5,958,341, now allowed, and is a continuation-in-part of U.S. Ser. No. 08/909,691 filed Aug. 12, 1997 now abandoned, the disclosures of which are incorporated fully herein by reference. 
    
    
     BACKGROUND OF THE INVENTION 
     This invention relates to an apparatus for processing biological samples on slides for a wide variety of purposes. Biological samples are analyzed for many purposes using a variety of different assays. Pathologists often use histochemistry or immunocytochemistry for analyzing biological samples, molecular biologists may perform in situ hybridization or in situ polymerase chain reactions on biological samples, etc. Often the sample to be analyzed will be embedded in paraffin and mounted on a microscope slide. 
     The assays usually involve the use of antibodies, enzymes and other expensive reagents and it is desirable to keep reagent volume use to a minimum to lower costs. These assays are also quite labor intensive although there are now some automated systems (e.g., the Ventana ESIHC Staining System, the Shandon Lipshaw Cadenza Automated Immunostainer; also see Brigati et al. (1988)). The publications and other materials used herein to illuminate the background of the invention or provide additional details respecting the practice, are incorporated by reference, and for convenience are respectively grouped in the appended List of References. Most automated systems can only perform 40 to 48 slides per run. Fisher automated systems can perform 120 slides per run. Most automated systems which only perform immunocytochemistry do not perform deparaffinizing, histochemistry (such as hematoxylin and eosin staining) and coverslipping steps and these consequently must be done separately by hand which is time and labor intensive. The automated systems perform only a small part of the overall process of preparing and analyzing slides. Steps which are still manually performed-prior to the automated portion include sorting of cases and slides, labeling slides, programming the automated equipment, daily antibody and reagent preparation, preparing control tissue which is mounted on slides, and microwave antigen retrieval. Procedures still performed manually after the automated steps are dehydration, coverslipping, slide labeling and sorting of slides and cases. Furthermore, most commercial ready-to-use reagents are not suitable for automated systems which are required to use specially designed reagents. Laboratories which process large numbers of samples are likely to be willing to pay the high cost associated with buying these automated systems as well as the high cost of using the disposable accessories and reagents to perform the assays, but small to intermediate sized laboratories find it more cost effective to continue to process samples manually. 
     A typical immunocytochemistry assay requires a series of many steps. These include: obtaining a biological sample such as from a biopsy, fixing the sample in formalin, processing the sample overnight, embedding the sample in paraffin, cutting serial sections and mounting on microscope slides and drying. These steps are followed by steps to deparaffinize (treatments in xylene, ethanol and water), and finally the reaction can be performed on the sample which has been mounted on the slide. Typically a series of solutions including reagents such as enzymes, primary antibody, secondary antibody, detection reagent, chromogen, counterstain, etc. is dropped onto the slide, incubated, and washed off. Finally the sample may be viewed under the microscope. Clearly there are many individual steps involved and each sample on a slide must be processed individually. Besides being very labor intensive, there are drawbacks associated with the commonly used method of simply dropping solutions on top of the mounted sample on the microscope slide. The solution is not restricted simply to the area of the biological sample itself and the solution may be relatively deep rather than being a thin layer. These features require use of extra reagents which are quite expensive. Leaving the solutions open to the air as they sit on the slide also may lead to evaporation if the samples must incubate for a long period of time. Evaporation leads to concentration or drying out of the reagents and high concentrations may lead to increased background levels which are clearly undesirable. If the solutions evaporate totally the assay will fail. Incubating samples in humidity chambers with covers may prevent evaporation problems, but water droplets which condense onto the humidity chamber cover may fall onto the slides and this will ruin the assay. 
     Improved methods for more rapidly assaying several samples at once, but without the high cost of automated systems, will be welcomed by small to intermediate sized laboratories. Furthermore, methods which will allow use of smaller amounts of reagents and overcome the drawbacks of processing samples on slides open to the atmosphere will be a welcome advance. 
     SUMMARY OF THE INVENTION 
     The present invention is an apparatus for performing manual assays on biological samples mounted on microscope slides. One aspect of the invention is a multislide slideholder capable of holding multiple standard microscope slides, preferably 3-10 slides and more preferably 3 or 6 slides, thereby allowing for the processing of multiple samples at one time. A second aspect of the invention is a multiwell tray containing multiple shallow wells, preferably 3-10 and more preferably 3 or 6 such wells, into which reagents are placed and upon which the slideholder plus slides is placed. A third aspect of the invention is a set of prealigned and prespaced coverslips for rapidly placing said coverslips onto the processed slides. Another aspect of the invention is a second type of multiwell tray which is useful in automating several of the steps of the procedures. 
     Besides this new design of a slideholder and corresponding tray and coverslips, other aspects of the invention are set out which aid in making assays more rapid and convenient. One such aspect is the use of reagents which are predried in the wells of the tray thereby simply necessitating the addition of water or buffer to the well without having to add the reagents at the time of assay. The well is then covered with a slide with a biological sample premounted on the slide. The different wells of a multiwell tray can be pretreated with different reagents dried in each well. Multistep assays can be performed by moving a slideholder with attached slides from one multiwell tray to the next, with each well of a multiwell tray having the desired reagents predried on it. A variation of this is to employ a multilayer coating of reagents in each well such that the first set of reagents dissolves quickly and acts upon the biological sample, the second layer then dissolves releasing the reagents for the second step, etc., thereby requiring the use of fewer trays, possibly only a single tray. 
     Another aspect of the invention is to have built in controls on each slide. This is a portion of the slide to which are attached positive and negative controls. These controls allow one to determine whether the assay has worked properly for each individual slide since each slide has its own set of controls and which simultaneously act as labels for each slide. 
     The slideholder is designed in conjunction with the tray. The purpose of the slideholder is to have multiple slides, preferably up to six slides, all attached to a single holder so that all the attached slides may be processed simultaneously throughout all of the steps of the staining procedure from deparaffinizing to coverslipping without ever separating the slides from the slideholder. This is a labor intensive step and the ability to process multiple slides at once rather than processing slides individually is an important aspect of the invention. Since one technician typically is capable of easily processing about 40-50 individual slides without mistakes, using a slideholder with six slides per slideholder will allow a single technician easily to perform approximately 240-300 slides for routine histochemistry and immunochemistry staining. This is about 2-6 times as many slides as handled by automated systems per each run. 
     One useful aspect of the present invention is that any kind of commercial ready-to-use reagents are compatible with the slideholder and there is no need to use specially designed reagents. Another useful aspect is that the apparatus allows a technician to perform different staining procedures on the different slides, e.g., histochemistry and immunocytochemistry or in situ hybridization and in situ PCR at the same time. Other important aspects of the present invention are that it allows one to observe chromogen color development as it is happening and it also gives results which have at least as low a background and sometimes a lower background level of staining than is seen in conventional techniques. 
     Clearly slideholders capable of holding more than six slides may be envisioned. However there are two practical reasons for utilizing holders capable of holding three or six slides. The first such reason is that the staining dishes in which slides are processed, e.g., washing of the slides, which are presently in use in the typical pathology lab are wide enough to handle only up to six slides side by side at a single time. Therefore a design to hold up to six slides will be compatible with presently used equipment. A second reason for designing a system for processing three or six slides at one time is that in a typical pathology assay three samples must be run together, these being the actual sample being assayed plus a known positive control plus a known negative control. Since a single assay typically requires the use of three slides a system capable of handling six slides allows for the processing of two patient samples at a time. 
     The slideholder may be designed in different ways. The purpose of the slideholder is to hold up to six microscope slides at one time so that one can easily manipulate the six slides simultaneously with one hand. In one embodiment a reusable slideholder is used. The reusable slideholder is designed to allow the tops of microscope slides to fit into the holder simply by inserting the slides into slots in the holder. The slides will fit firmly so as not to fall out during handling. The holder positions the slides in the slots so that the slides are prealigned to fit onto the wells of the tray containing the reagents which will react with the biological samples. The thickness of the holder is designed so that it fits into a trough in the tray and keeps the microscope slides level on top of the wells. If the holder is too thick or too thin the slides may not rest properly on top of the wells in the lower tray and proper contact will not be made with the reagents in the wells. 
     The slideholder need not be a reusable one. In this second embodiment of the invention one end portion of each microscope slide is glued to a slideholder using a glue which is xylene and ethyl alcohol resistant. The glue holds the slides firmly to the slideholder during processing and when the processing, e.g., staining, is completed the slides can be easily separated from the slideholder. 
     A third embodiment of the invention is a slideholder with suction cups attached wherein said suction cups hold the slides firmly on the slideholder. 
     A fourth embodiment of a slideholder is one in which the slide holder consists of two plastic portions with ridges wherein one portion is placed onto each side of the slides and then clipped together such that the slides are held between the two portions of the slideholder. The ridges properly align and space the slides. In one variation of this and other embodiments, the slideholder has ribbed surfaces of plastic or rubber which help to hold the slides firmly in place. 
     In any embodiment of a slideholder it is useful to have a handle portion to make the handling of the slideholder simpler, but the presence of a handle is not essential. If a handle is present it is useful to have holes in the handle through which a “fork” can be inserted such that several (15-20) slideholders can be placed onto a single fork and manipulated together easily. Another useful variation that is applicable to any embodiment of slideholder is to have an opening or a transparent region of the slideholder which acts as a window through which one can see a label attached to the end of the slide which is inserted into the slideholder. Yet another desirable feature which may be used is to have slideholders of various colors which make it simple to determine which slides are undergoing which assay when a variety of assays is being performed. 
     The tray to contain the reagents such as antibodies and enzymes is an integral part of some embodiments of the invention. The tray is preferably designed with three or six wells although trays with a different number of wells may also be used. In one embodiment of the present invention, each well is shallow to hold a minimal amount of reagent to keep costs low but is deep enough to allow for fluid motion within the well. This overcomes some problems present with systems requiring capillary action of fluid between two slides, especially when viscous reagents must be used. See, Babbitt et al., U.S. Pat. No. 5,002,736; D. J. Brigati, U.S. Pat. No. 4,777,020; Bowman et al., U.S. Pat. No. 4,985,206; and McGrath et al., U.S. Pat. No. 5,192,503. Each well is completely separated from neighboring wells by a trough so that any overflow from one well cannot contaminate a neighboring well. A tray may be designed without these intervening troughs if one is not concerned about contamination between wells, e.g., if all of the wells contain the same solution. The microscope slides with mounted biological samples are placed sample side down on top of the wells of the tray and completely cover the wells effectively sealing the wells from the atmosphere. This aspect of the invention prevents evaporation of the small amount of fluid in the well. It further prevents contamination of the reagents in the well and also overcomes the problem of extraneous matter falling on top of the sample such as sometimes occurs when samples are incubated in a covered humidity chamber and drops of water fall onto the surface of the slides. In the present invention any such drops of water fall onto the backs of the incubating slides since the slides are placed sample side down onto the trays. 
     Another embodiment of the tray is one in which the bottom of each well is made of a soft or pliable material. One advantage to having a soft bottom is that it becomes easy to remove air bubbles which may be trapped between the slide and soft bottom. By pushing on the soft bottom of a well, one can easily remove air bubbles to a region away from the region of the biological sample. A second advantage to having a soft bottom is that the volume of reagent solution needed in a well becomes flexible. 
     A related embodiment is the use of a tray with a flexible bottom. The bottom may be soft or it may be a hard material which is capable of being moved. This movement can be as simple as applying pressure to a solid plastic tray bottom to make it flex or it can be more complex such as a hinged bottom. The purpose of such a flexible bottom is that the volume within a well can be adjusted. This is useful because if one is performing a reaction, e.g., PCR, in a very small volume of about 10-15 μL in the well, it is very difficult to pump this small volume out of the well because of the force of the capillary action of the small amount of liquid between the tray and the covering slide with biological sample. To overcome this, the well volume can be made small to encompass only the 10-15 μL volume when desired following which the volume can be increased to accommodate more fluid so that the fluid can be easily pumped through the well and collected if desired. 
     Another feature of the tray is two notches or channels (which act as ports or vents) in the well boundaries and tubing attached to the outside of the boundary at one of these channels. These features allow the slide holder plus slides to be placed onto the tray prior to adding reagents to the tray. The reagent solutions may be added through the tubing. A simple way of adding solutions between the slides and the wells of the tray is to immerse the tray with slideholder and slides vertically into the solutions. Expensive reagents can be pipetted directly through a notch or port. The solutions pass from the tubing through a first channel or port or while air in the well escapes through a second channel (a vent) at the top of the boundary. 
     Yet another possible feature of a tray is a channel through which reagents can be added. Such a channel can be centrally located as shown by feature  430  in FIG.  16 B. Such a channel can extend from the absolute base of the tray through the base and into a well region of the tray. Effectively this means that each well has a hole in its bottom. If desired a second channel or hole can be included in the bottom of each well to act as a vent for air to escape as reagent is added through the first channel. 
     Other alternate embodiments of the tray may be used but will not necessarily have all of the advantages outlined above. One such alternate embodiment is to design a tray with larger wells such that each well can accommodate multiple slides at one time, preferably 3 or 6. In this design slides are placed into a holder which holds the slides in tight conjunction side by side. In a design with a tray containing wells large enough to require 3 slides to completely cover each well, a slideholder will hold 3 slides in tight conjunction (or 2 sets of 3 slides with a space between the 2 sets). With this configuration it is necessary to make only a single pipetting to fill a well for 3 slides rather than requiring 3 individual pipetting steps for the 3 slides. This is a labor saving advantage. The disadvantage is that the volume of a single well to be covered by 3 slides is greater than 3 times the volume of a well designed to be covered by an individual slide. This is because wells designed for single slides are narrower than the full width of a slide. This alternate embodiment is therefore a tradeoff of requiring fewer pipettings thus saving time vs. the added expense of using slightly more reagent. Furthermore, these larger wells may only be used when each slide is to be treated with the identical reagent. These larger wells are also useful in the cases for which large biological samples are examined with the single sample requiring several slides side by side for mounting. Trays with individual wells are suited to treating each slide with different reagents since each well is completely separate from nearby wells. 
     Another desirable feature which may be used with any embodiment of tray is to have trays of various colors which make it simple to determine which slides are undergoing which assay when a variety of assays is being performed. 
     A different embodiment of the invention is that rather than a tray, a special multichamber coverslip is placed on top of a slide which has a biological sample mounted on top of it. This special coverslip consists of three or six conjoined incubation chambers. A further feature of this special coverslip is that the top of each incubation chamber comprises a soft and pliable top rather than simply being a hard coverslip. The purpose of the soft top is to be able to push any trapped air bubbles to a region away from the biological sample. Another feature is that the special coverslip can include a raised region toward the edges of each chamber which can trap air which is pushed into the region and thus trap air bubbles which have been pushed to the edges thereby preventing the air bubbles from returning to the area of the slide on which the biological sample is mounted. Another advantage of the soft top is that the volume of reagent solution needed in a chamber becomes flexible. 
     An alternative embodiment of the special coverslip is to modify it to have tubing on one side of the chamber and a very small hole on the opposite side of the chamber. The tubing may contain a valve through which reagents can be added or removed and by which means the tubing can be closed. The small hole allows air to come out when reagent is added to the chamber. This modification allows the coverslip to be placed onto the slides prior to adding reagent, with reagent later being added via the tubing. Another desirable feature which may be used with any embodiment of coverslip is to have coverslips of various colors which make it simple to determine which slides are undergoing which assay when a variety of assays is being performed. 
     The third aspect of the invention is a set of slide covers which are premade as a set of multiple covers, preferably six covers, connected together and which may be laid on top of the processed slides which are still attached to the slideholder. The dimensions are such that all covers will perfectly line up on the set of slides. The covers are then easily detached from the holder. This may be accomplished by simply breaking them off by having a pre-scored region which allows for easily snapping off the coverslip from a “holder” region to which the slides are attached. The ability to simultaneously align and mount up to six slide covers is a time saving technique which is useful in such a labor intensive process. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The file of this patent contains at least one drawing executed in color. Copies of this patent with color drawings will be provided by the Patent and Trademark Office upon request and payment of the necessary fee. 
     FIG. 1A is a front elevational view of a slideholder  1  showing the front planar surface  10  and the groove  20  which is 1 cm wide, runs the complete length from edge  30  to edge  32  and is 0.4 cm from edge  34  and 2.6 cm from edge  36 . 
     FIG. 1B is a rear elevational view of a slideholder  1  showing the opposing planar surface  12  and two sets of six grooves  40  and  42 . Regions  50  bound a side of each slide  70  and form the edges of each slot. Region  60  is a 0.6 cm wide strip extending from edge  30  to edge  32 . 
     FIG. 1C is a view of a slideholder looking end on at edge  34  showing slots  56 . Front planar surface  10 , opposing planar surface  12 , boundary regions  50 , and edges  30  and  32  are also indicated. 
     FIG. 1D shows a slideholder  1  with a handle  5 . The handle  5  has holes  11  through which a fork  100  (not shown) may be inserted. The slideholder  1  has openings  7  through which may be seen labels which are on the slides  70 . Position  15  is a space on the slideholder  1  to which a label may be attached. Slides  70  are inserted into slot  56 . 
     FIG. 1E is similar to FIG. 1D but indicates the presence of slides  70  inserted into slots  56 . 
     FIG. 1F is similar to FIG. 1D but shows a simpler slideholder  1  which does not include openings  7 . 
     FIG. 1G is a cross-sectional view of the slideholder shown in FIG.  1 D. This shows a slide  70  partially inserted into slot  56 . Opening  7  is indicated. This indicates an embodiment of a slideholder which has ribbed surfaces  9  within slot  56 . 
     FIG. 2A is an elevational view of one surface of a slideholder  1 . Slides  70  are attached to the slideholder  1  by gluing an end of slide  70  into an indentation  22  between ridges  6 . 
     FIG. 2B shows an edge view of the slideholder  1  shown in FIG.  2 A. 
     FIG. 3A is a front elevational view of one surface  3  of one portion of a third embodiment of a slideholder  1 . There are seven raised ridges  6  which align and space the slides  70  parallel to each other and a ridge  8  against which one end of the inserted slide is pushed. 
     FIG. 3B is a rear elevational view of the third embodiment of a slideholder  1  as shown in FIG.  3 A. This opposing surface  4  is a flat planar surface. 
     FIG. 3C is similar to FIG. 3A except it shows a slideholder  1  with a window  7  through which one can read a label attached to a slide  70 . The Figure also illustrates a handle  5 . 
     FIG. 3D is similar to FIG. 3B except it shows a slideholder  1  with a window  7  through which one can read a label attached to a slide  70 . The Figure also illustrates a handle  5 . 
     FIG. 3E is a cross sectional view of slideholder  1  taken along line  74 — 74  of FIG.  3 A. This shows a slideholder  1  which has ribbed surfaces  9 . These are not shown in FIG. 3A which is a design not including the ribbed surfaces  9 . 
     FIG. 3F is a cross sectional view of slideholder  1  taken along line  74 — 74  of FIG.  3 A. This shows a slideholder  1  which has ribbed surfaces  9  wherein the ribbed surfaces are of a sawtooth pattern. These are not shown in FIG. 3A which is a design not including the ribbed surfaces  9 . 
     FIG. 4A is a front elevational view of a tray  14 . Wells  24  are separated by troughs  38 . Boundaries  44  of wells  24  are flat and are elevated above the interior portion of the wells  24 . 
     FIG. 4B is a cross sectional view of the tray  14  taken along line  54 — 54  of FIG.  4 A. This view shows wells  24 , troughs  38 , and well boundaries  44 . Slide  70  is shown resting on one well  24 . 
     FIG. 4C is a cross sectional view of the tray  14  taken along line  64 — 64  of FIG.  4 A. This shows the hook region  66  which aids in keeping slides  70  pressed against the tray  14 . 
     FIG. 4D shows a soft-bottomed tray  14 . Wells  24 , troughs  38  and well boundaries  44  are indicated. A notch or channel  45 , to allow air to escape, in the boundary  44  is shown. Tubing  146  through which to apply solution to the well  24  via channel  47  is shown. Tubing  146  includes a valve  149 . 
     FIG. 4E is a cross sectional view of the tray  14  along line  54 — 54  taken through all six wells  24  shown in FIG. 4D. A curved bottom of wells  24  indicates that these are soft bottomed wells. Troughs  38  and well boundaries  44  are shown. A slide  70  is shown resting on top of one well  24 . 
     FIG. 4F is a cross sectional view of the tray  14  along line  64 — 64 . This illustrates hook region  66 . 
     FIG. 5 shows coverslips  18  and indicates scoring at scoreline  80 . 
     FIG. 6 illustrates a fork  100  and the alignment of tines  110  of fork  100  with holes  11  in the handle  5  of slideholder  1 . 
     FIG. 7A illustrates a top elevational view of special multiple chamber coverslip  140 . The shaded regions  160  are ridges which extend down between slides and on the edges of the outermost slides  70 . Region  150  is a raised region of the coverslip  140  into which air may be pushed and trapped. A small hole  148  through which air may escape is indicated. A tubular opening  146  through which solution may be pipetted is indicated. This opening  146  may contain a valve  149  which seals the opening. 
     FIG. 7B is a cross sectional view of special multiple chamber coverslip  140  as viewed along line  154 — 154  in FIG.  7 A. This illustrates the raised ridges  150  which create a space into which air may be pushed and trapped. Also seen are the ridges  160  which fit between and around slides  70  and align the coverslip  140 . Region  144  which lies in a rectangle inside of raised ridges  150  is made of a soft material. 
     FIG. 7C is an enlarged view of tubular opening  146  and valve  149  which were shown in FIG.  7 A. 
     FIG. 7D is a cross sectional view of special multiple chamber coverslip  140  as viewed along line  155 — 155  in FIG.  7 A. This shows a slide  70  held between ridges  160 , and raised ridges  150  within ridges  160 . Slot  152  is the region between ridges  160 . This is more clearly seen in FIG.  7 E. Tubular opening  146  is also indicated. 
     FIG. 7E shows a bottom elevational view of special multiple chamber coverslip  140 . Slides  70  which are held by slideholder  1  are inserted into slots  152  of coverslip  140 . 
     FIG. 8A shows a top elevational view of an incubation coverslip  170  suitable for performing in situ PCR. This coverslip  170  consists of a stiff region  176  surrounding a softer region  174 . The example shown is one wherein three coverslips are joined together to simultaneously process three samples, these being a positive control, a negative control, and the experimental sample. A framework  182  supports the soft top  174 . A heat sealable tubular opening  180  for adding solution may be included. 
     FIG. 8B shows a cross sectional view along line  178  of FIG.  8 A. This shows the coverslip  170  sitting on a slide  70  and illustrates the soft top region  174  surrounded by the stiffer region  176 . Also shown are a tube  180  in the soft top region  174  and a pipet tip  190 . 
     FIG. 9A shows a slideholder  1  which uses suction cups  200  to hold slides  70  to the slideholder  1 . The suction cup  200  is shown with an optional tab  210  which is lifted to break the vacuum between the suction cup  200  and the slide  70 . Although circular suction cups  200  are illustrated, other shapes are within the scope of the invention. 
     FIG. 9B shows an edge view of the slideholder  1  shown in FIG.  9 A. The rightmost slot shows a suction cup  200  prior to being depressed by slide  70 . The slot to the left of this shows the suction cup  200  depressed by slide  70  which is thereby affixed to the slideholder  1 . 
     FIGS. 10A-D show normal lymph nodes that were formalin-fixed overnight at room temperature and immunohistochemically stained with CD20 (L26) monoclonal antibody at a 1:250 dilution. FIGS. 10A and 10C show results when CD20 antibody was dropped onto the slides for staining via the commonly used procedure. These show a magnification of 30× and 150× respectively. FIGS. 10B and 10D show the results when the staining was performed using the present invention. These show a magnification of 30× and 150× respectively. Here the CD20 was dropped into the wells of the tray. Note the increased intensity of immunostain with decreased background staining in the sample which was processed using the present invention. 
     FIGS. 11A-D show a normal lymph node that was formalin-fixed overnight at room temperature and immunohistochemically stained for immunoglobulin kappa light chains polyclonal antibody 1:25,000 dilution. FIGS. 11A and 11C show the results using the standard method of dropping antibody directly on the slide. These show a magnification of 75× and 150× respectively. This resulted in a high background staining. FIGS. 11B and 11D show results when the staining was performed using the tray assembly of the present invention and dropping solution into the tray. These show a magnification of 75× and 150× respectively. This resulted in a lower background level of staining with overall stronger staining. 
     FIGS. 12A-D show a normal lymph node that was formalin-fixed overnight at room temperature and immunohistochemically stained for immunoglobulin lambda light chains polyclonal antibody 1:50,000 dilution. FIGS. 12A and 12C show the results using the standard method of dropping antibody directly on the slide. These show a magnification of 75× and 150×, respectively, and show the resulting high background staining. FIGS. 12B and 12D show results when the staining was performed using the tray assembly of the present invention and dropping solution into the tray. These show a magnification of 75× and 150× respectively. These show a lower background level of staining with overall stronger staining. 
     FIG. 13 illustrates a slide  70  with a biological sample  220  and a stamp  230 . The stamp shown contains reagents A-F. 
     FIG. 14 illustrates a well  24  in which three reagents (indicated as  250 ,  260 , and  270 ) have been dried and onto which has been placed a slide  70  with mounted biological sample  220 . Layers of inert material separating the layers of reagents from each other are not shown. 
     FIGS. 15A-B illustrate one well of a multiwell tray  330  which is used to automate several steps of the procedure of assaying a biological sample in conjunction with a thermal cycler, pumps and a central processing unit. FIG. 15A shows slide  70  with mounted biological sample  220  placed on a well or reaction chamber  280 . Inlets  300  and  302  and outlets  294  and  296  which connect to reaction chamber  280  are illustrated. The portion of tray  330  which forms the bottom of the reaction chamber  280  is shown as  282 . Optional stops  281  are shown which prevent the reaction chamber bottom  282  from pressing up against sample  220 . The view in FIG. 15A shows the reaction chamber bottom  282  in an “open” mode which causes the reaction chamber  280  to have a large volume. FIG. 15B shows the tray and slide of FIG. 15A in conjunction with other optional equipment. In FIG. 15B the reaction chamber bottom  282  is in a “closed” mode such that reaction chamber  280  encompasses a smaller volume than seen in FIG.  15 A. Piston  284  to move reaction chamber bottom  282  is shown. The piston  284  is controlled by central processing unit  286 . A thermal cycler  288  is illustrated pressed against slide  70 . The thermal cycler can also be controlled by central processing unit  286 . Tubing can be attached to the inlets  300  and  302  and to the outlets  294  and  296 . Pumps  290  attached to the tubing are shown and pump liquid to or from reservoirs  291  or  292  or to gel  298 . 
     FIGS. 16A-E illustrate a tray used to perform whole chromosome painting of multiple chromosomes on cells on a single slide or which can be used to perform in situ hybridization or FISH on a biological sample. FIG. 16A illustrates an 8 well tray  400  with wells  410 . Each well is separated from neighboring wells by troughs  420 . Each well  410  has an opening or channel  430  through which liquid can be pipetted. FIG. 16B is a side view of the 8 well tray  400  shown in FIG. 16A. A slide  70  is shown on the tray  400 . Four wells  410  are illustrated with three of the wells being empty and one shown filled with liquid. Openings  430  and troughs  420  are also illustrated. FIG. 16C is an end-on view of the slide and tray of FIGS. 16A and 16B. Trough  420  is shown between two wells  410 . Openings  430  into the wells  410  are shown. Slide  70  is shown resting above sides of tray  400  showing optional clips  402  to hold slide  70  to tray  400 . FIG. 16D is a schematic showing a slide  70  illustrating 8 regions  440  of the slide which will be in contact with each of the 8 wells  410 . This is only illustrative, there being no need to actually denote these regions  440  on the slides used in practice. FIG. 16E illustrates one manner of designing built-in controls on slide  70  by showing an enlargement of one region  440 . Each region  440  has nucleic acids  442 , which hybridize to the probes being used in the assay, placed in an array around the perimeter of region  440 . These controls will be in contact with probe during the hybridization. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The present invention is an integrated system for manually processing biological samples on microscope slides in a more rapid and efficient and less costly manner than is typical. By a biological sample is meant a tissue section, biopsy, cell smear, nucleic acid, protein or peptide, chromosome, bodily fluid or other biological material commonly observed under a microscope. The system consists of a slideholder  1  and a tray  14  or a coverslip  140  for simultaneously holding multiple, preferably up to six, microscope slides  70  to allow for concurrent processing of the multiple slides  70 . The slideholder  1  may be reusable. Some embodiments of slideholders are shown in FIGS. 1-3. The holder  1  must not be so thick that it is thicker than the trough  90  in which it sits in the well-containing tray  14 . If the holder  1  is too thick the microscope slides  70  will not lie flat on top of the wells  24  and there will be poor contact with the reagents inside of the wells  24 . If the holder  1  is thinner than the trough  90  in which it sits there may be a problem in that if the holder  1  is too heavy it will fall to the bottom of the trough  90  and cause the slides  70  to angle up above the wells  24  and result in poor contact with the reagents in the wells  24 . If the holder  1  is not so heavy then the weight of the slides  70  will cause them to remain flat on top of the wells  24  and the fact that the holder  1  is thinner than the depth of the trough  90  will be of no consequence. A different solution to this problem is to have a hook  66  which grabs the top of one edge of a slide  70  and holds the slide against the well boundary  44  thereby preventing the slideholder from falling into trough  90 . This helps to ensure that the biological sample on all slides  70  will make good contact with the reagents in the wells  24 . 
     One embodiment of a slideholder is shown with reference to FIGS. 1A-1C. The slideholder  1  may be made of a stiff plastic material such as polyethylene, polypropylene or polycarbonate or any of the other suitable plastics which are xylene and alcohol resistant and which are well-known to those in the art. An example of suitable dimensions for the slideholder  1  is 17.5 cm×4 cm. The slideholder  1  preferably contains 6 slots  56  2.6 cm (slightly larger than the width of a standard slide) by 0.1 cm (the thickness of a standard slide) into which slides  70  are inserted. Each slot  56  is deep enough to allow approximately the top 1.9 cm of each slide  70  to be inserted. Each slot  56  is separated from a neighboring slot  56  by 0.3 cm. This slideholder  1  will hold the slides  70  firmly in place and properly align and space the slides  70  to fit the exemplary tray  14  described above. 
     The slideholder  1  may be machined from a rectangular planar piece of a rigid plastic material of dimensions 17.5×4×0.3 cm. The front planar surface  10  is machined to carve out a groove  20  1 cm in width and very slightly less than 0.2 cm in depth traversing the full 17.5 cm length of the holder  1 . Groove  20  is at a distance 2.6 cm from edge  36  of the holder  1  and 0.4 cm from edge  34  of the holder  1 . The back or opposing planar surface  12  of the holder  1  is machined to carve out two sets of six grooves  40  and  42 . The first set of 6 grooves consists of grooves  42  2.6 cm long and 0.65 cm wide said width being measured from edge  34  of said holder  1  toward edge  36  of said holder  1 . Each groove  42  is made very slightly less than 0.2 cm in depth from the opposing face  12  toward the front face  10 . Each groove  42  is separated from any neighboring groove  42  or from a side edge of the holder  1  by an approximately 0.3 cm region  50  of plastic which is not machined thus leaving said regions  50  0.3 cm in depth from the front planar surface  10  to the opposing planar surface  12 . A second set of 6 grooves  40  is machined into the opposing planar surface  12  such that there is a distance of 0.6 cm between edge  46  of the set of 6 grooves  40  and edge  48  of the set of 6 grooves  42 . The set of grooves  40  exactly aligns with the set of grooves  42  with each groove  40  being 2.6 cm long, 0.65 cm wide and very slightly less than 0.2 cm in depth with unmachined regions  50  of 0.3 cm between each groove  40 . The holder  1  which results from inserting groove  20  across the front planar surface  10  and the two sets of grooves  40  and  42  into the opposing planar surface  12  is a holder consisting of 6 slots  56  which are partially enclosed on both surfaces and into which can be inserted approximately 1.9 cm of each of 6 slides  70 . The slides  70  are held firmly in each slot  56  by the tension of the plastic surfaces but the slides  70  are easily removable by gently pulling on them. 
     The above description is only one example of a slideholder which may be used for the present invention and is not meant to be limiting. Many differently designed slideholders may be envisioned which may be made with different dimensions or even in quite different manners. The slideholder need not be manufactured in the manner described above but may be made by a molding process, a different machining process, or other methods well-known to those of skill in the art. Slideholders such as shown in FIGS. 1D-1F may be prepared. 
     A second embodiment of a slideholder  1  is shown in FIG.  2  and is simply a rectangular strip of material, preferably plastic, with indentations  22  on one face to which 6 slides  70  are attached by some means such as a glue which is xylene and alcohol resistant applied to region  22  such that the slides  70  may be easily removed from the rectangular strip. The slides  70  are attached such that there is a 0.3 cm gap between neighboring slides  70 . These dimensions will properly align the slides  70  to fit into the tray  14  mentioned above. 
     FIGS. 3A and 3B illustrate a third embodiment of a slideholder  1 . In this embodiment the slideholder  1  comprises two plastic pieces  2  which are held together by a binder clip or by glue. Either one or both pieces  2  of this third embodiment have substantially parallel ridges  6  on one surface  3 , said ridges  6  being 2.5 cm apart with each ridge  6  0.3 cm wide. The other surface  4  of this slideholder  1  is a flat surface. The combined height of opposing ridges  6  on the two pieces  2  is less than the depth of a slide  70 , this generally being 0.1 cm. These ridges  6  align the slides  70  properly and allow the plastic pieces  2  to firmly hold the slides  70  in place when slides  70  are placed between them and a clip is placed on the plastic pieces  2  to hold them together or alternatively the pieces  2  are glued together. The clip must fit properly into trough  90  of tray  14  to allow the slides  70  to lie flat on wells  24 . In a preferred embodiment both pieces  2  of this embodiment of the slideholder  1  are identical. It is not necessary that the two pieces  2  be identical, for example one piece  2  could have ridges  6  and the second piece  2  could be flat with no ridges  6 , the ridges  6  on a single piece  2  being enough to properly align the slides  70 . A preferred embodiment further has a ridge  8  against which the top edge of each slide  70  is pushed so that an equal length of each slide  70  is protruding from the slideholder  1 . 
     FIGS. 3C and 3D illustrate an alternative design which incorporates an opening  7  in the slideholder  1  through which it is possible to read a label attached to a region of the slide  70  which is inserted into the slideholder  1 . This opening  7  may be present in either one or both pieces of the slideholder  1 . 
     Other variations of the above embodiments are possible. The first embodiment described above may be formed by using two pieces of plastic which are later sealed together rather than machining a single piece of plastic to form the slideholder  1 . In such a case and in a slideholder  1  such as the third embodiment described above, it is possible to attach ribbed surfaces  9  of plastic or rubber or to machine ribs into the slideholder  1 . FIGS. 1G,  3 E and  3 F illustrate these ribbed surfaces  9 . The ribbed surfaces  9  aid in preventing an inserted slide  70  from slipping out of the slideholder  1 . 
     Yet another embodiment of a slideholder  1  is one wherein slides  70  are attached to slideholder  1  by means of suction cups  200  which are mounted on slideholder  1 . Such an embodiment is illustrated in FIG.  9 . Suction cups  200  may comprise a tab  210  which when pulled releases the vacuum between the suction cup  200  and a slide  70 . 
     Another aspect of the invention is to color code the slideholder and the handle of the slideholder. For each different procedure a different color of slideholder and handle may be used. For example, the handle color may be used to indicate whether the sample is for histochemical staining, immunocytochemistry, in-situ PCR, etc. The slideholder in turn can have its own color which may be different from or the same as the color of the handle. The slideholder may even consist of multiple colors. The slideholder color can be indicative of information, e.g., to indicate whether a sample has or has not been digested, is being treated with a monoclonal or a polyclonal antibody, etc. The color coding scheme is a matter of personal choice. Coding schemes other than color can be used, such as numbers, letters or other symbols, but a color scheme is preferred. 
     The slideholder  1  plus slides  70  is placed on top of a multiple well tray  14  which preferably contains up to six individual wells  24  or 1 or 2 large wells  24  with each large well  24  to be covered by 6 or 3 slides  70  respectively. FIG. 4A is a representation of one embodiment of the tray  14 . An example of the dimensions of such a tray  14  is (see FIG.  4 ): outer dimensions of tray  14  19×11 cm; outer dimensions of each well  24  of 2.2×4.7 cm, inner dimensions of each well  24  1.8×4.3 cm (therefore leaving a flat edge  44  0.2 cm wide surrounding each well  24  and upon which each slide  70  will lie); a space or trough  38  between each well  24  of 0.3 cm. Each well  24  is raised above the height of the trough  38  by approximately 0.1 cm with the edges  44  surrounding the well  24  being approximately 0.1-0.3 mm above the center region of the well  24 . These values are exemplary only and are not meant to limit the invention. The listed values are appropriate for standard sized slides (25×75 mm), allow for using small amounts or reagents, and space the slides  70  closely enough that 6 slides  70  will fit within the width of a standard staining dish. The type of material from which the tray  14  is made will depend on the type of assay being performed. For many purposes the tray  14  may be made of a thin, moldable plastic material. It may be desirable to use a clear, transparent material so that wells  24  can be viewed from beneath. Such trays  14  are easily manufactured and may be used once and disposed. If the tray  14  is to be subjected to high temperatures such as occurs with polymerase chain reactions, it will be more appropriate to manufacture the tray  14  of aluminum which is capable both of withstanding the high temperatures to which it will be exposed and of efficiently conducting heat which is a necessity for the polymerase chain reaction to work properly. 
     In a preferred embodiment each well  24  is separated from the neighboring well  24  by a trough  38 . This trough  38  prevents cross-contamination between wells  24 . The depth of each well  24  is approximately 0.1 to 0.3 mm and will hold approximately 75-200 μL of solution. Trays  14  with wells  24  of different depths may be desirable for specific types of reactions. Deeper wells  24  (on the order of 0.3 mm) may be used when it is desirable to have a larger amount of reagent present and yet prevent the necessity of having the reagent very concentrated. Conversely a tray  14  with shallower wells  24  may be used when a smaller amount of reagent is adequate for the desired purpose. The use of smaller amounts of expensive reagents is one of the advantages of the present invention. As can be seen in FIG. 4A, the tray  14  consists of three or six wells  24  with each well  24  surrounded by a trough  38 . The trough  38  is extended to include trough  90  into which the slideholder  1  can fit. This area is necessary for a reusable slideholder  1  to allow the microscope slides  70  to lay flat on top of the wells  24 . The thickness of the holder  1  underneath the slides  70  must be no greater than the depth of the trough  90 . 
     In practice, a biological sample is mounted onto each of the slides  70  to be analyzed. This often involves steps of fixing a biological sample in formalin, embedding the sample in paraffin, cutting thin, serial sections from the paraffin, and mounting the sections onto the microscope slides  70 . These are dried overnight at room temperature. The mounted biological samples are subjected to some type of assay such as staining. For this the mounted samples must be placed in contact with a series of solutions with washing steps in between each different change of reagent. In the present invention the reagents are measured into each well  24  in the trays  14 . Enough reagent is added to completely fill the well  24  such that the solution in the well  24  will contact the microscope slide  70  which is to be laid on top of the well  24 . There should be no air bubbles present between the solution in the well  24  and the microscope slide  70 . By exactly filling the well  24  or by slightly overfilling the well  24  so that there is a slight overflow once the slide  70  is placed on top of the well  24  (surface tension holding the top of the solution in the well  24  prior to a slide  70  being placed onto it) there is no problem with air bubbles forming. Capillary action of the fluid in the well  24  contacting the slide  70  allows for good contact between the biological sample and reagents across the complete well  24  area and helps to seal the well  24 . Trays  14  may be designed to include a hook  66  on one edge of a well boundary  44 . This is shown in FIGS. 4C and 4F. By pushing all of the slides  70  against the hooks  66  all of the slides will be held against the well boundaries  44  and this will assure good contact with the reagents within the wells  24 . 
     By placing the slides  70  onto the tray  14  in the above manner, the mounted biological sample is facing down into the well  24  and is not exposed to the atmosphere. This prevents extraneous material from falling into the reagent or onto the biological sample during incubation. Furthermore, the slide  70  covers the well  24  and helps to prevent evaporation of the reagent solution in the well  24  during incubation. Evaporation may lead to very bad background signals. The present invention helps to overcome this problem. 
     After incubation with each reagent the slideholder  1  and tray  14  are picked up and put into a standard staining dish with 500 milliliters of phosphate buffered saline (PBS) solution. Once in the PBS, the surface tension between the slides  70  and the tray  14  disappears and the slides are very easily removed from the tray. The slides are then put through the appropriate washing steps. It is a simple matter to pick up six slides  70  at once since they are all attached to a single holder  1 . A standard staining dish in a laboratory is large enough to accommodate six slides  70  across (as attached to a single slideholder  1 ) and can contain 20 slideholders  1 . Therefore 120 slides  70  may be washed and processed simultaneously. If slideholders  1  with handles  5  containing holes  11  in them are utilized, it is very convenient to slide the tines  110  of a single fork  100  through the holes  11  of several slideholders  1 , even up to at least 20 slideholders  1 , at one time. This is illustrated in FIG.  6 . The dashed lines  130  in FIG. 6 indicate how the tines  110  fit through the holes  11  of the slideholder  1 . All of the slideholders  1  are then picked up and moved between staining dishes simply by picking up a single fork  100 . The fork  100  may have a handle  120  for ease of use. In the third embodiment of slideholder  1  described above which used a clip or glue to hold together two pieces of the slideholder  1 , the clip acts as a handle  5  and may be made to have holes  11  through which the tines  110  of fork  100  may be placed. If glue is to be used, the slideholder  1  may be designed with a handle  120  so that no clip is necessary. 
     Following the processing of a sample it is customary to place a coverslip  18  onto each slide  70 . This has customarily been done one slide  70  at a time. The present invention makes this chore easier by having premanufactured coverslips  76  which are connected preferably in groups of three or six and which are spaced to properly line up with the three or six slides  70  in the slideholder  1 . Six coverslips  18  may be picked up at once and aligned over three or six slides  70  simultaneously. The coverslips  18  are usually a thin piece of glass or plastic. These may be manufactured to be prescored so that each individual coverslip  18  easily snaps off from its holder  78 . FIG. 5 shows a diagram of one method of connecting six coverslips  18  and showing how each group of six is scored along line  80  for easy separation. 
     Another embodiment of tray  14  is one in which the bottom of each well  24  is made of a soft or pliable material. The purpose of the soft bottom is that it becomes easy to remove air bubbles which may be trapped under a slide  70 . By pushing on the soft bottom of a well  24  one can easily move air bubbles to a region away from the region of the biological sample. In this embodiment it may be especially desirable to make the wells  24  from a transparent material to make it simpler to observe any air bubbles which may be present. Aside from the well  24  bottoms it is best to manufacture the remainder of the tray  14  from a stiff material for ease of handling. The advantage of the soft bottom is that the necessary volume of reagent solution to be added to a well becomes flexible. 
     Another embodiment of tray  14  is a notch or channel  45  on the top of well boundary  44  and a channel  47  in the bottom of well boundary  44 . A tubing  146  is on the outside of the bottom well boundary  44 . Tubing  146  includes a valve  149 . This embodiment allows the slideholder  1  plus slides  70  to be placed onto wells  24  of tray  14  prior to adding solution to the wells  24 . The solution can be added later through tubing  146 . The solution enters wells  24  from tubing  146  via channel  47 . Air that is in wells  24  escapes through notch or channel  45  as solution is added. Solution can also be removed through tubing  146 . 
     A different embodiment of the invention is one in which no tray  14  is used, rather the biological samples are mounted onto slides  70 , the slides  70  are left face up, and a special multichamber coverslip  140  is placed on top of the slides  70 . This is illustrated in FIGS. 7A and 7B. It is preferred that the slides  70  are first placed into a slideholder  1 . The special coverslip  140  actually consists preferably of three or six conjoined coverslips  142  properly spaced so as to align with slides  70  which are in a slideholder  1 . A further feature of this special coverslip  140  is that it comprises “soft tops”  144  rather than simply being a hard coverslip. The purpose of the soft top  144  is to be able to push any trapped air bubbles to a region away from the biological sample. Again, it is desirable to manufacture these from a transparent material such that it is easier to observe trapped air bubbles. Another feature is that these special coverslips  140  may have a raised region  150  toward the edges of each coverslip  142  which can trap air which is pushed into the region  150  and thus trap air bubbles which have been pushed to the edges and thereby prevent the air bubble from returning to the area of the slide  70  on which the biological sample is mounted. FIGS. 7A-E illustrate this special multichamber coverslip. The soft top  144  is in the region between the raised region  150 . Although soft top  144  is illustrated as a rectangular area in FIG. 7B it can be any other desired shape such as an oval or circle. Aside from the soft top  144  the rest of the special coverslip  140  is made of a stiff material. Ridges  160  are present to easily align the coverslip  140  properly onto slides  70 . 
     Another feature which may be included in this multichamber coverslip  140  is to include slots  152  into which the ends of slides  70  may be inserted. Thus one end of each slide  70  will be inserted into slideholder  1  and the opposite end will be inserted into a slot  152  of coverslip  140 . This slot  152  will help to align and hold the coverslip  140  on slides  70  during transportation of slideholder  1 , slides  70  and coverslip  140 . 
     An additional feature which can be included in this embodiment is to include a tubing  146  on one side of the coverslip  140  and a very small hole  148  on the other side. The tubing  146  is connected to a valve  149  through which reagents can be added and which can be closed to seal the tubing  146 . This feature allows the multichamber coverslip  140  to be placed onto the slides  70  of the slideholder  1  prior to addition of solutions. The solutions are then added through tubing  146 . The air in the chamber can escape through the very small hole  148 . 
     A variation of this last embodiment is a specially designed coverslip  170  to be used for in situ PCR. This is illustrated in FIGS. 8A and 8B. FIG. 8A is an elevational view which shows three coverslips joined together. This coverslip  170  has a “soft top”  174 , e.g., polyethylene or low density polyethylene, which allows for expansion and contraction of the PCR reaction fluid on the biological sample during the temperature cycling. The soft top region  174  is surrounded by a stiff region  176  which is outside the region of the biological sample. In its most simple form, the PCR solution is placed onto the biological sample on a slide  70  and the coverslip  170  is placed onto the slide  70  such that the soft top region  174  is over the biological sample. Stiff region  176  may be adhesive backed and will stick onto the slide  70  and seal the coverslip  170  onto the slide  70  and prevent evaporation. This soft top bubble type incubation coverslip works like a balloon. When the temperature increases, it will expand and when the temperature decreases it will shrink in response to the expansion and contraction of the liquid within the well. The pressure inside the well chamber will be significantly reduced by this soft top design. This low pressure may reduce or eliminate the expansion and contraction of the solution and allow mainly only an up and down movement of the solution thereby restraining the movement of newly formed products from their original sites. Consequently, this inhibits the diffusion of PCR products and increases the signal at the original sites. 
     The soft top bubble type incubation coverslip looks like an umbrella or a tent with a high fixed frame and shape to prevent the soft top coverslip from touching the biological sample on the slide. This is illustrated in FIG.  8 B. However, the soft top has enough space to expand and contract without generating a high pressure. This design advantage allows the use of regular plastic material and eliminates the need for using steel clips and a silicone disc to prevent leaking. 
     As illustrated in FIG. 8A one can join three coverslips  170  together to simultaneously process positive and negative controls along with the experimental sample. These may be designed to cover three biological samples all mounted onto a single slide if desired. By placing all three samples on a single slide the PCR is more consistent across all three samples. 
     PCR coverslip  170  may be modified to perform hot start PCR. This is illustrated in FIG.  8 B. For this the soft top  174  is modified to be made of a stiffer plastic material, e.g., polypropylene, and to include a short tube  180  through which reagents may be added. The coverslip is placed onto the slide  70  with the soft top covering the biological sample. The first portion of the PCR solution is pipetted through the tube  180 . The slide is placed onto a thermal cycler and heated. Following the initial heating the remaining reagents are added by pipetting through tube  180 . Tube  180  may then be sealed with a heat sealer. This prevents evaporation of fluid during the cycling steps. 
     Another aspect of the invention is to predry reagents in wells  24  of trays  14  thereby requiring simply the immersion of the tray  14  and slides  70  into water or buffer or the pipetting of water or a buffer into the wells  24  at the time of assay. Trays  14  can be prepared which include a series of reagents predried in the wells  24  of a multiwell tray  14 , e.g., each well  24  of a multiwell tray  14  can have a different set of reagents dried in the well  24 . At the time of assay, slides  70  can have a biological sample from a single patient or from different patients mounted on them and be placed onto a single tray  14  to perform multiple assays at once. Such trays  14  with predried reagents can be prepared ahead of time and stored until the time of use. As currently practiced, assays performed on biological samples are performed by fixing a sample onto a slide and then dropping reagents onto the sample. Such a method cannot take advantage of premeasured, predried reagents which require only the addition of water or buffer. In the invention disclosed here, the reagents can be predried in a well  24  on a tray  14 , buffer or water is added to well  24 , and a slide  70  with biological sample mounted on it is placed on top of well  24 , sample side down. The buffer or water may be added to well  24  via tubing  146  after placing slide  70  on top of well  24 . Having slide  70  over well  24  forms a sealed reaction chamber which prevents contamination and evaporation and also ensures uniform distribution of reagents as compared to dropping solution on top of a slide as is generally done in current practice. 
     Yet another aspect of the invention is to have built-in controls and/or labels on each slide. Known controls are immobilized onto each slide in a region apart from the biological sample. For example, the controls can be antigens, peptides, proteins or cells which are being tested for in the biological sample or can be a nucleic acid of known sequence if a hybridization assay is being performed. These would act as positive controls which should give a signal or color if the assay works properly. Negative controls can also be placed onto the slide, e.g., a protein or antigen or a nucleic acid which should not react with the reagents in the well. For example, assume a person is to be tested for the presence of six antigenic determinants A-F. A six well tray can be used with each well containing a different antibody A′-F′. The six different antigenic determinants can be spotted onto all six slides. In all cases, only a single one of these controls should show as positive on each slide. Slide A should show only antigenic determinant A as a positive signal, slide B should show only antigenic determinant B as a positive signal, etc. These act as external controls. If more than one control shows as a positive, this indicates antibody cross reaction has occurred. If none of the controls is positive it indicates that the reaction did not work, e.g., a reagent may have been missing. The biological sample being tested acts as an internal control. 
     The external controls can be placed onto each slide by a variety of means. A preferred mode is to spot the reagents onto the equivalent of a postage stamp or sticker, which uses glue resistant to xylene and alcohol, which can then be glued onto each slide. Such a stamp or sticker can be made of any suitable material to which proteins, peptides, cells or nucleic acids bind tightly. This can include, but is not limited to, commonly used membranes such as nitrocellulose, plastic, glass or nylon. Specific examples of such membranous material are nitrocellulose itself, Immobilon-P (Millipore), Hybond-N, Hybond-N +  and Hybond C-extra nitrocellulose (Amersham), Genescreen and Genescreen Plus (Du Pont), Clearblot-P (ATTO Co.) and polyvinyldifluoride membranes (Millipore or BioRad). The stamp or sticker will have regions A-F as shown in FIG.  13 . These stamps or stickers can be premanufactured and stored until ready for use, the antigenic determinants, proteins, peptides, cells or nucleic acids being dried onto the stamps or stickers. The name of the antigen, protein, cell, etc., can be printed on the stamp or sticker. This is especially suitable for mass production. Standard sets of assays can be premade such as a panel to test for breast cancer or a panel to test for Hodgkin&#39;s disease, but one can always design any combination of reagents as external controls as are desired. A stamp of controls can be attached to a slide either prior to a biological sample being placed upon the slide or it may be delayed until the biological sample has been fixed on a slide and been processed to the point at which reactions relevant to the controls are to be performed. 
     The stamps can be color coded or numbered to indicate a specific panel of tests to be performed. In like fashion the tray  14  can be color coded or numbered or otherwise marked to indicate the panel of tests to be performed, this being dependent upon the predried reagents in the wells  24  of the tray  14 . The stamp and the tray should match colors or numbers or other marking. 
     One other aspect of the invention is that reagents which are dried in wells  24  can be dried in layers in the reverse order which they are to act. When buffer is added the last added reagent will dissolve first and be active, followed by the next to last added reagent which acts in turn, etc. In this manner two or more reagents can be added to a single well  24  thereby allowing consecutive action of the reagents without the necessity of moving the slides  70  from one tray  14  to a second tray  14 . For multistep reactions this will decrease the number of trays  14  which are necessary and also decreases the amount of labor involved. 
     Another aspect of the invention is a specially designed tray or chip which allows one to perform whole chromosome painting of all 24 human chromosomes on cells on a single slide. 
     Still another aspect of the invention is a tray and slide assembly wherein the volume of space in the well of the tray can be adjusted so that a small volume can be present to perform a reaction such as a PCR and then the volume of space can be increased to allow fluid to be pumped through the well. 
     Those of skill in the art recognize that the sample to be tested on the slide including the protein, peptide, DNA, RNA or cells or the control protein, peptide, DNA, RNA or cells on the stamp, must be immobilized so that they will not be released during the assay. The reagents which may have been predried in the tray, however, which reagents may include proteins, peptides, nucleic acids, etc., should be released, in a programmed order if multilayered, once the water or buffer has been added. 
     EXAMPLES 
     In each example a biological sample is first mounted onto a microscope slide  70  and then assayed. Surgical and autopsy human biological samples from various organs (lymph node, liver, kidney, lung, breast, skin, prostate) were routinely fixed in 10% neutral buffered formalin, processed overnight on a tissue processor, and embedded in paraffin. Serial sections are cut at 4-5 microns and mounted onto Probe-On-Plus Slides (#15-188-52; Fisher Scientific) and dried overnight at room temperature. Slides  70  are then inserted into a reusable slideholder  1 . At this point all the slides  70  in a single holder  1  (up to six slides) can be handled simultaneously. The slides  70  are deparaffinized by placing the slides  70  in a staining dish with four changes of xylene for 5 minutes each, two treatments of 100% ethanol for 1 minute each and two treatments of 95% ethanol for 1 minute each. The deparaffinized tissue section slides  70  are cleared and washed with deionized water. 
     The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the invention in any manner. Standard techniques well known in the art or the techniques specifically described below are utilized. 
     Example 1 
     Hematoxylin and Eosin (H &amp; E) 
     Hematoxylin and eosin is the most common staining procedure used in pathology. Every case must have H &amp; E staining for making a pathologic diagnosis. Deparaffinized tissue section slides  70  which are in slideholders  1  are placed vertically into a staining dish with 500 mL of hematoxylin solution for two minutes followed by washing with running tap water in a staining dish for five minutes. The slides are placed in 95% ethyl alcohol for one minute and counterstained in eosin-phloxine solution for two minutes. The samples are dehydrated and cleared using two changes each of 95% ethyl alcohol, absolute ethyl alcohol, and xylene for two minutes each. 
     Coverslips are attached as follows: Place one drop of Cytoseal 60 or premount on the tissue section side of each slide  70  with the slides  70  still attached to the slideholder  1 . Place coverslips  18  onto each slide  70 . Although this may be done one by one, it is more efficient to use a specially designed coverslip  76  which is actually six (or three) conjoined coverslips  18  properly spaced to align with six (or three) slides  70 . Using this special coverslip  76 , up to six individual coverslips  18  are effectively aligned and placed onto slides  70  simultaneously. The coverslips  18  are easily separated from the plastic strip  78  holding them together simply by bending the coverslip  76  which is prescored to allow the strip  78  to snap apart from the coverslips  18  which remain bound to the slides  70 . At this point the slides  70  may be removed from the slideholder  1  to be handled individually, or they may be left attached to the slideholder  1  for ease of transportation. 
     Example 2 
     Immunocytochemistry 
     In this Example a biological sample is treated with antibodies (primary and secondary), treated for chromogen color development, and finally counterstained. 
     A. Proteolytic Pretreatment of Mounted Tissue Samples 
     It is well known in the art that when using certain antibodies for immunocytochemical staining it is necessary to pretreat the formalin fixed tissue section with proteolytic enzymes such as 0.4% pepsin, pH 2.0. When this is necessary the following steps may be utilized. A few drops (150-200 μL) of the proteolytic digestion solution are placed on each well  24  of the 3 or 6 well tray  14 . The tissue side of the slides  70  is faced down on the wells  24 . The slideholder  1  with the slides  70  should be slowly laid down and placed on the wells  24  of the tray  14 . No air bubbles should remain between the tissue side of the slides  70  and the solution in the wells  24  of the tray  14 . The slides  70 , slideholder  1  and tray  14  with solution are incubated for 15 minutes at 40° C. 
     If many samples are being processed at one time it is more efficient to forgo use of the tray  14  during this proteolytic pretreatment step. The slides  70  are still placed into slideholders  1  six to a holder  1 . The slideholders  1  and slides  70  are then placed vertically into a staining dish with 500 mL of the proteolytic digestion solution (which may be reused) and incubated for 20 minutes at 40° C. in a water bath. Up to twenty slideholders  1  (120 slides) may be simultaneously placed into the staining dish for this pretreatment step. 
     Some antibodies require that the tissue section be pretreated with microwave antigen retrieval. Slideholders  1  (up to 20) with slides  70  are vertically placed into a staining dish with 500 mL of 0.01 M citrate buffer, the staining dish is placed in the center of a microwave oven, and the oven is turned to high power (800-850 Watts) for 7-8 minutes bringing the solution to a rapid boil. The oven is turned off, the power level is reset to 400 Watts, and the oven is turned on again to heat the solution for 7-8 minutes. 
     After proteolytic digestion and microwave treatment the tissue sections are washed in the staining dish with three 500 mL changes of phosphate buffered saline (PBS). 
     B. Treatment of Tissue Sections With Goat and Horse Serum 
     All slides  70 , whether or not proteolytically digested and microwave treated, are incubated with 5% mixed normal goat and horse serum for 20-30 minutes at room temperature. Each well  24  of a tray  14  is filled (approximately 150-200 μL) with mixed normal goat and horse serum. The tissue side of the slides  70  is placed down on the wells  24  to contact the serum. The slideholder  1  should be slowly laid down so as to avoid trapping any air between the slides  70  and the wells  24 . Again, if many samples are being processed at one time, it is more efficient to perform this step as a batch by placing up to 20 slideholders  1  vertically into a staining dish with 500 mL of 5% mixed normal goat and horse serum for 20-30 minutes. 
     C. Application of the Primary Antisera or Antibodies 
     Following incubation with the serum, the slideholder  1  and slides  70  as well as the tray  14  are put into a staining dish with PBS. The tray  14  is separated from the slideholder  1  and both are washed once with PBS. The washed tray  14  may be reused for the next step. Prediluted primary antisera or antibodies (approximately 150-200 μL) are applied to each well  24  of the tray  14 . The washed slides  70 , still in the slideholder  1 , are placed tissue side down onto the wells  24 . As always care must be taken to avoid trapping bubbles between the slide  70  and the reagent solution in the wells  24 . The samples are incubated with the antisera or antibodies for 2-4 hours at room temperature or incubated in a humidity chamber at 40° C. for 2 hours or may be incubated in a humidity chamber at room temperature overnight. After incubation the slideholder  1  and attached slides  70  are removed from the tray  14  and are washed in a staining dish with PBS three times. 
     D. Application of the Secondary Antibody 
     Prediluted secondary antibody (approximately 150-200 μL) is applied into each well  24  of a new tray  14 . The slides  70  in the slideholder  1  are placed onto the wells  24  tissue side down being careful to avoid bubbles. This is incubated for 30 minutes at 40° C. in a humidity chamber. After incubation the slideholders  1  and attached slides  70  are removed from the tray  14  and washed in a staining dish with three changes of PBS. 
     E. Treatment for Removal of Endogenous Peroxidase Activity 
     All slideholders  1  with attached slides  70  are placed into a staining dish with 500 mL of PBS with 3% hydrogen peroxide and 0.1% sodium azide, and incubated at room temperature for 15 minutes. After incubation with the hydrogen peroxide PBS the slideholders  1  and attached slides  70  are washed in a staining dish with three changes of PBS. 
     F. Application of the ABC Complex “ELITE” 
     The ABC complex (Vector Laboratories Inc., Burlingame, Calif.) is diluted to its working concentration using PBS. The working concentration (approximately 150-200 μL) is applied to each well  24  of a new tray  14 . The slides  70  with attached slideholders  1  are carefully placed tissue side down onto the trays  14  so that no air bubbles are trapped between the solution and the slides  70 . The slides  70  and trays  14  with ABC solution are incubated in the humidity chamber at 40° C. for 30 minutes. After incubation the slideholders  1  with attached slides  70  are removed from the trays  14  and washed in a staining dish with 3 changes of PBS. 
     G. Chromogen Color Development Using Diaminobenzidine (DAB) 
     DAB solution is prepared by adding 100 mg DAB to 100 mL PBS and adding 50 μL of 30% H 2 O 2 . Approximately 150-200 μL of the DAB solution is added to each well  24  of a new tray  14  to completely fill each well  24 . The slides  70  with attached slideholders  1  are placed tissue side down onto the wells  24  being careful to avoid trapping air bubbles. Color development can be monitored by viewing the slideholders  1  and trays  14  with DAB under a microscope. A colored precipitate will form at the site of positive cells. Color begins to appear after 2-5 minutes, usually reaching sufficient development within 10 minutes, but a 20-30 minute incubation may be necessary for weakly stained samples. To stop development, all slideholders  1  with slides  70  are removed from the trays  14  and washed in a staining dish with three changes of deionized water. 
     H. Counterstaining 
     Slideholders  1  and attached slides  70  are immersed in Harris&#39;s hematoxylin for 10-50 seconds and washed by dipping into deionized water for three changes. Then all the slides  70  are immersed in 0.2% ammonium hydroxide solution for 30 seconds and washed by dipping in deionized water for 3 changes. The slides  70  are dipped into 95% ethanol for two changes of 2 minutes each, followed by dipping into 100% ethanol for 2 changes of 2 minutes each, and finally the slides  70  are cleared by dipping into two changes of xylene for 2 minutes each. 
     I. Attachment of the Coverslip 
     Place 1 drop of Cytoseal 60 or premount on the tissue section side of each slide  70  with the slides  70  still attached to the slideholder  1 . Place coverslips  18  onto each slide  70 . Although this may be done one by one, it is more efficient to use a specially designed coverslip  76  which is actually six (or three) conjoined coverslips  18  properly spaced to align with six (or three) slides  70 . Using this special coverslip  76 , up to 6 individual coverslips  18  are effectively aligned and placed onto slides  70  simultaneously. The coverslips  18  are easily separated from the plastic strip  78  holding them together simply by bending the coverslip  76  which is prescored to allow the strip  78  to snap apart from the coverslips  18  which remain bound to the slides  70 . At this point the slides  70  may be removed from the slideholder  1  to be handled individually, or they may be left attached to the slideholder  1  for ease of transportation . 
     FIGS. 10-12 show the results of a study comparing the use of the present invention with staining methods simply using the standard manual method of dropping reagents onto the surface of a slide-mounted tissue sample and leaving the reagents open to the atmosphere for incubation. The Figures show that the results obtained with the two methods are extremely comparable with the results obtained using the present invention being at least as good as, and apparently better than, the results obtained using the traditional method. The present invention however allowed these results to be obtained with less work and with the use of smaller amounts of reagents. 
     Comparing the two methods, the background staining is significantly reduced by using the present invention, especially when using polyclonal antibodies (anti-kappa light chain antibodies and anti-lambda light chain antibodies). The invention significantly improves the staining results by reducing the background. Background is partially due to free FC fragments which precipitate by gravity and bind nonspecifically to the tissue. The present method inverts the slide such that the tissue is above the solution and therefore free FC fragments cannot precipitate by gravity onto the tissue. 
     Example 3 
     In situ Hybridization 
     In this example biological samples are mounted onto slides  70 , hybridized with biotin or digoxigenin labeled probes and reacted with anti-biotin or anti-digoxigenin antibody. The samples are then stained. 
     A. Preparation and Mounting of Tissue Sample 
     A tissue sample is prepared as described above but with extra measures to prevent nucleic acid degradation. A tissue sample is fixed in 10% neutral buffered formalin, processed overnight on a tissue processor, embedded in paraffin, cut into serial sections of 4-5 microns, mounted onto Probe-On-Plus Slides (#15-188-52; Fisher Scientific), and dried overnight at room temperature. The slides  70  are inserted into a slideholder  1  and are deparaffinized by placing into a staining dish. The slides  70  are treated with four changes of xylene for 5 minutes each, two changes of 100% ethanol for 1 minute each and two changes of 95% ethanol for 1 minute each. The deparaffinized tissue section slides are then cleared and washed with deionized water with RNase Block (BioGenex, San Ramon, Calif.). 
     B. Proteinase K Treatment of the Mounted Tissue Samples 
     Approximately 150-200 μL of freshly diluted proteinase K solution is placed into each well  24  of a tray  14  to completely fill each well  24 . The microscope slides  70  (still in the slideholder  1 ) are placed onto the wells  24  with the tissue side down. The slides  70  are placed onto the wells  24  carefully so as to avoid the presence of air bubbles between the solution in the wells  24  and the slide  70 . This is incubated for 15 minutes at room temperature. 
     After digestion, the slideholders  1  with slides  70  attached are removed from the tray  14  and washed in a staining dish with 500 mL of PBS with RNase Block for 5 minutes. The tissue section slides  70  are dehydrated by immersing in a staining dish serially in the following solutions: 500 mL distilled water plus RNase Block for 10 seconds, 500 mL 50% ethanol plus RNase Block for 10 seconds, 500 mL of 95% ethanol for 10 seconds, and 500 mL 100% ethanol for 10 seconds. The slides  70  are dried at room temperature for 5 minutes. 
     C. Hybridization With Biotinylated or Digoxigenin Labeled Probes 
     Trays  14  with shallow wells  24  (0.02-0.08 mm in depth) may be used here to conserve materials. Hybridization solution containing a biotinylated or digoxigenin labeled oligonucleotide probe is placed into each well  24  of a tray  14 . Enough solution is added to each well  24  to completely fill the well  24 . This requires approximately 50-100 μL of solution. The slides  70  are placed on top of the wells  24  (3 or 6 at a time still attached to the slideholders  1 ) being careful not to trap any air bubbles. The trays  14  plus slideholders  1  and slides  70  are placed in an oven or on a heating block at 95° C. for 8-10 minutes to denature the nucleic acids. This step eliminates hair-pin loops or folding back of mRNA sequences. After the denaturation step, the slides  70  are incubated in a humidity chamber at 45° C. overnight. Following the hybridization step, the slides  70  are washed by removing the slideholders  1  with attached slides  70  from the trays  14  and washing the slides  70  in a staining dish with 2×SSC (standard saline citrate) at 37° C. for 5 minutes followed by a wash with 1×SSC at 37° C. for 5 minutes. This is followed by a 30 minute wash in 0.2×SSC at 60° C. Finally the slides  70  are washed with 2 changes of PBS for 2-5 minutes each. 
     D. Signal Detection 
     The slideholders  1  with attached slides  70  are placed vertically into a staining dish with 500 mL of 5% mixed normal goat and horse serum at room temperature for 20 minutes. Prediluted mouse anti-biotin or mouse anti-digoxigenin antibody (150-200 μL) is applied to each well  24  of a new tray  14 . The slides  70  are placed onto the wells  24  of the tray  14  taking care to avoid trapping bubbles. The slides  70  and trays  14  with antibody are incubated in a humidity chamber at 40° C. for 2 hours. 
     After incubation with the anti-biotin or anti-digoxigenin antibody, the slideholders  1  with slides  70  are removed from the trays  14  and washed in a staining dish with three changes of PBS. 
     E. Application of the Secondary Antibody 
     Prediluted secondary antibody (approximately 150-200 μL) is applied into each well  24  of a new tray  14 . The slides  70  in the slideholder  1  are placed onto the wells  24  tissue side down being careful to avoid bubbles. This is incubated for 30 minutes at 40° C. in a humidity chamber. After incubation the slideholders  1  and attached slides  70  are removed from the tray  14  and washed in a staining dish with three changes of PBS. 
     F. Treatment for Removal of Endogenous Peroxidase Activity 
     All slideholders  1  with attached slides  70  are placed into a staining dish with 500 mL of PBS with 3% hydrogen peroxide and 0.1% sodium azide, and incubated at room temperature for 15 minutes. After incubation with the hydrogen peroxide PBS the slideholders  1  and attached slides  70  are washed in a staining dish with three changes of PBS. 
     G. Application of the ABC Complex “ELITE” 
     The ABC complex is diluted to its working concentration using PBS. The working concentration (approximately 150-200 μL) is applied to each well  24  of a new tray  14 . The slides  70  with attached slideholders  1  are carefully placed tissue side down onto the trays  14  so that no air bubbles are trapped between the solution and the slides  70 . The slides  70  and trays  14  with ABC solution are incubated in the humidity chamber at 40° C. for 30 minutes. After incubation the slideholders  1  with attached slides  70  are removed from the trays  14  and washed in a staining dish with 3 changes of PBS. 
     H. Chromogen Color Development Using Diaminobenzidine (DAB) 
     DAB solution is prepared by adding 100 mg DAB to 100 mL PBS and adding 50 μL of 30% H 2 O 2 . Approximately 150-200 μL of the DAB solution is added to each well  24  of a new tray  14  to completely fill each well  24 . The slides  70  with attached slideholders  1  are placed tissue side down onto the wells  24  being careful to avoid trapping air bubbles. Color development can be monitored by viewing the slideholders  1  and trays  14  with DAB under a microscope. A colored precipitate will form at the site of positive cells. Color begins to appear after 2-5 minutes, usually reaching sufficient development within 10 minutes, but a 20-30 minute incubation may be necessary for weakly stained samples. To stop development, all slideholders  1  with slides  70  are removed from the trays  14  and washed in a staining dish with three changes of deionized water. 
     I. Counterstaining 
     Slideholders  1  and attached slides  70  are immersed in Harris&#39;s hematoxylin for 10-50 seconds and washed by dipping into deionized water for three changes. All the slides  70  are immersed in 0.2% ammonium hydroxide solution for 30 seconds and washed by dipping in deionized water for 3 changes. The slides  70  are then dipped into 95% ethanol for two changes of 2 minutes each, followed by dipping into 100% ethanol for 2 changes of 2 minutes each, and finally the slides  70  are cleared by dipping into two changes of xylene for 2 minutes each. 
     J. Coverslipping 
     Place 1 drop of Cytoseal 60 or premount on the tissue section side of each slide  70  with the slides  70  still attached to the slideholder  1 . Place coverslips  18  onto each slide  70 . Although this may be done one by one, it is more efficient to use a specially designed coverslip  76  which is actually six (or three) conjoined coverslips  18  properly spaced to all line up with six (or three) slides  70 . Using this special coverslip  76 , up to 6 individual coverslips  18  are effectively aligned and placed onto slides  70  simultaneously. The coverslips  18  are easily separated from the plastic strip  78  holding them together simply by bending the strip  78  which is prescored to allow the strip  78  to snap apart from the coverslips  18  which remain bound to the slides  70 . At this point the slides  70  may be removed from the slideholder  1  to be handled individually, or they may be left attached to the slideholder  1  for ease of transportation. 
     Example 4 
     PCR in situ Hybridization 
     Polymerase chain reaction (PCR) was developed as an in vitro method for amplifying small amounts of specific pieces of nucleic acids. This was later adapted to in situ studies so that there was amplification of nucleic acid within tissue sections. The apparatus of the present invention is suited to performing these in situ PCRs. An example of a PCR in situ hybridization protocol is given in Nuovo (1994). 
     A. In situ PCR 
     Serial tissue sections are cut at 4-5 microns thickness, mounted onto Probe-On-Plus slides  70 , and dried overnight at room temperature. The mounted tissue sections are deparaffinized and digested with pepsin at 40° C. for 15-90 minutes depending on the length of time of fixation in formalin. The pepsin is inactivated by washing the slides  70  in diethylpyrocarbonate (DEPC) treated water for one minute followed by a one minute wash in 100% ethanol. The slides  70  are then air dried. 
     Polymerase chain reaction solutions are made according to any standard procedure. See, e.g., K. B. Mullis et al., U.S. Pat. No. 4,800,159. Combine buffer, 5′ and 3′ primers, water, Taq polymerase (AmpliTaq, Perkin Elmer) (or other thermophilic polymerase) and Self-Seal Reagent (MJ Research, Inc.) in a total volume of 20-50 μL. Apply the 20-50 μL of solution to a well  24  of a specially designed in situ PCR aluminum tray  14 . The trays  14  to be used in Examples 1 and 2 are preferably made of a disposable plastic material, but the trays  14  used for PCR studies must be capable of being cycled through a series of temperatures which may reach 95-100° C. Therefore it is necessary for such trays  14  to be heat resistant (i.e., they should not melt or otherwise be destroyed by high temperatures) and also to be good conductors of heat. Aluminum is a preferred material from which to manufacture these trays  14 . These aluminum trays  14  have wells  24  which are 0.005-0.03 mm in depth and hold approximately 20-50 μL of solution. 
     After completely filling each well  24  of the aluminum tray  14 , the slideholder  1  and attached slides  70  are placed on top of the tray  14  with the tissue section facing down so as to contact the solution in the well  24  upon which it is placed. Care must be taken to avoid air bubbles being present between the solution and the slide. The slideholder  1 , slides  70  and aluminum tray  14  are then placed onto a block of a thermal cycler at 95° C. for 3-5 minutes to denature the nucleic acids in the tissue. Twenty to thirty cycles are then performed cycling between 60° C. for 2 minutes and 94° C. for 1 minute. 
     Following the cycling steps, the slideholder  1 , slides  70  and aluminum tray  14  are placed vertically into a staining dish with 2×SSC at 37° C. for 5 minutes. The slideholder  1  is removed from the aluminum tray  14  and washed with 0.5-1×SSC at 37-60° C. for 10-30 minutes (depending upon background). In situ hybridization is performed as described in Example 2 using a biotinylated or digoxigenin labeled probe chosen internal to the primers. 
     B. Reverse Transcriptase in situ PCR 
     Serial tissue sections are cut at 4-5 microns thickness, mounted onto Probe-On-Plus slides  70 , and dried overnight at room temperature. An important aspect of the RT in situ PCR is that both negative and positive controls be performed and it is preferred that these be performed on the same glass slide. The positive control omits the DNAse digestion step and should generate an intense nuclear signal from target specific amplification, DNA repair and mispriming. The negative control uses a DNAse treatment plus primers that do not correspond to a target in the cells. The test sample undergoes DNAse treatment but uses primers specific to the desired target nucleic acid. The mounted tissue sections are deparaffinized and digested with pepsin at 40° C. for 15-90 minutes depending on the length of time of fixation in formalin. The pepsin is inactivated by washing the slides  70  in diethylpyrocarbonate (DEPC) treated water for one minute followed by a one minute wash in 100% ethanol. The slides  70  are then air dried. 
     Digest two of the three mounted tissue sections with RNase-free DNAse by filling each well  24  of a plastic tray  14  (requiring approximately 150-200 μL) with prediluted RNase-free DNAse and placing the slides  70  (in the slideholder  1 ) tissue side down on top of the well  24  being careful that air bubbles are not trapped and that contact is made between the solution in the well  24  and the tissue sample. Incubate overnight at 37° C. Inactivate the RNase-free DNAse with a 1 minute wash in DEPC water and a 1 minute wash in 100% ethanol. Let the slides  70  air dry. 
     The reverse transcription is performed using the EZ RT PCR system (Perkin Elmer). The RT/amplifying (RT-PCR) solution contains EZ rTth buffer, 200 μM each of dATP, dCTP, dGTP and dTTP, 400 Hg/mL bovine serum albumin, 40 Units RNasin, 0.8 μM of 5′ and 3′ primers, 2.5 mM manganese chloride, 5 Units of rTth, and 2× concentrated Self-Seal Reagent (MJ Research, Inc.). Twenty to fifty μL of the RT-PCR mixture is placed into each of three wells  24  in a specially designed in situ PCR aluminum tray  14  (the depth of the wells  24  is approximately 0.005-0.03 mm) to fill the wells  24 . The slides  70  are carefully placed onto the wells  24  with the tissue being placed in contact with the solution inside of the well  24 . The slides  70 , slideholder  1  and aluminum tray  14  are placed onto a block of a thermal cycler at 65° C. for 30 minutes followed by a denaturation step at 94° C. for 3 minutes. Twenty to 30 cycles are performed, each cycle being 60° C. for 2 minutes followed by 94° C. for 1 minute. 
     Following the cycling steps, the slideholder  1 , slides  70  and aluminum tray  14  are placed vertically into a staining dish with 2×SSC at 37° C. for 5 minutes. The slideholder  1  is separated from the aluminum tray  14  and washed with 0.5-1×SSC at 37-60° C. for 10-30 minutes (depending upon background). In situ hybridization is performed as described in Example 2 using a biotinylated or digoxigenin labeled probe chosen internal to the primers. 
     Those of skill in the art recognize that amplification schemes other than PCR are now well known and widely used and can be used in place of PCR. These include ligation amplification (or ligase chain reaction, LCR) and amplification methods based on the use of Q-beta replicase. Also useful are strand displacement amplification (SDA), thermnophilic SDA, and nucleic acid sequence based amplification (3SR or NASBA). See, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202 and Innis et al. (1990) for PCR; Wu and Wallace (1989) for LCR; U.S. Pat. Nos. 5,270,184 and 5,455,166 and Walker et al. (1992) for SDA; Spargo et al. (1996) for thermophilic SDA and U.S. Pat. No. 5,409,818, Fahy et al. (1991) and Compton (1991) for 3SR and NASBA. 
     Example 5 
     Wells With Multilayered Dried Reagents 
     Assays can be performed with a single reagent predried in a well  24  and if the use of several reagents is required, the slide  70  with biological sample can be moved from a first well  24  with the first reagent to a second well  24  with the second reagent, etc., wherein the various wells  24  can either be on the same or on separate trays  14 . Alternatively, more than one reagent may be predried in a well  24 . The reagents can be dried in layers with the outermost layer being the first reagent to be used. This is demonstrated in FIG. 14 which shows a slide  70  with cells or tissue section  220  placed over a well  24  into which has been predried in order: a secondary antibody  270 , a primary antibody  260 , and a protein blocking reagent  250 . In this manner, different reagents are separated and dry stored thereby preventing reaction until the addition of water or buffer to the well. Upon addition of water (if salts are predried in the well) or buffer to the well, the protein blocking agent  250  will dissolve first since it was in the final layer of reagents predried in the well  24 . Next the primary antibody  260  will dissolve and finally the secondary antibody  270  will dissolve and be able to react. Such a system allows all three steps to occur without the necessity of moving the slides  70  from one tray  14  to another tray  14  or from one well  24  to another well  24 . For a different type of assay, for example one which requires a series of four reagents, one may either predry all four reagents in reverse order of action in a single well  24  or it may be found that the use of two trays each with two reagents or one tray with three reagents and a second tray with either the first or fourth reagent works better, for example when a wash step is needed between the step or steps of the first tray and the step or steps of the second tray. Other variations on these schemes are obvious to one of skill in the art. Any such combination requires less manual labor then the use of four separate trays. Especially in the field of pathology for which the types of assays to be performed are well standardized, such a system is quite amenable to mass production of trays with predried reagents which can then be stored until time of use. This system is not limited to the use of antigen/antibody reactions but can also be used for other reactions, e.g., enzymes can be dried in the wells, nucleic acid hybridization can be performed with different probes dried in the wells, a fluorescent probe can be the dried reagent, biotin can be dried in the well, etc. 
     To prepare wells with multiple layers of different reagents, it is preferred to include layers of inert material between the layers of reagents. For example, a well may be coated with reagents as follows. A secondary antibody is coated onto a well and allowed to dry. On top of this is coated a high concentration of an inert material (i.e., a material not necessary for any of the reactions and which will not interfere with the reactions) such as bovine serum albumin, gelatin, sucrose, fetal calf serum, starch, agarose or other inert material. This is allowed to dry. It is preferred that the inert material be added in several layers, e.g., gelatin in solution is added, allowed to dry, then more gelatin in solution is added, allowed to dry, etc. This can be performed as often as desired, the number of layers affecting the delay time until the release of the secondary antibody. Five such coatings on top of the secondary antibody has been found to give good results with a delay of about 15-20 minutes until the release of the secondary antibody from the time this inert layer begins to dissolve. On top of this first layer (or multilayer) of inert material is coated a primary antibody which is allowed to dry. On top of the primary antibody is coated a second layer or multilayer of inert material. This can be a low concentration of bovine serum albumin, gelatin, fetal calf serum, starch, agarose or other inert material. Three coatings of this second inert layer has been found to yield good results with a delay time of about 10 minutes until the release of the primary from the time the second inert layer begins to dissolve. On top of the second inert layer is coated a protein block such as horse and goat serum. The protein block is allowed to air dry. The multilayers of inert material take time to dissolve thereby giving each reaction enough time to occur prior to the next layer of active reagent dissolving. 
     The limitation of this system is that it can only be used for a series of steps which do not require a wash step in between successive steps. For example, if reaction with a primary antibody is followed by reaction with a secondary antibody, the secondary antibody must be washed off prior to the detection step. Therefore the detection reagent cannot be predried in the same well as the secondary antibody. Similarly, if one step requires heating (e.g., denaturation of a nucleic acid probe) this cannot be combined with a reagent which is heat inactivated or destroyed. 
     Example 6 
     Built-in Controls and Automatic Labels—Immunoassays or Ish/fish 
     When assays are performed in a clinical setting, controls are required by the Food and Drug Administration. Having built-in controls on the very slides being assayed is an excellent manner in which to test the controls. If the control is on a completely different slide, the control is not as good because it cannot indicate whether there was a problem such as reagent not contacting the biological sample on either the control or the actual test sample or missing a step of adding a reagent to either the control or the test sample. Also, the reagents dropped onto the control sample may accidentally be different from those dropped onto the test sample by a human or by machine error, especially when several tests are being performed simultaneously. When the control is on the same slide as the test sample, such problems will be indicated by controls, but if the control is a section of normal or neoplastic tissue it is very labor intensive and time consuming to prepare the control sample. 
     FIG. 13 illustrates a slide  70  onto which a tissue slice  220  has been fixed and also illustrates a separate region of slide  70  onto which has been affixed a stamp or sticker  230  (e.g., a piece of nitrocellulose or other membrane or plastic or glass type matrix glued onto the slide  70 ) with six distinct regions A-F, although the use of a stamp or sticker is not essential, e.g., the controls can be directly coated onto the slide  70 . Each region of A-F has been spotted with, e.g., a distinct antigenic substance or nucleic acid, depending on the type of assay being performed, although these substances can be applied directly to a region of the slide  70  in lieu of using a stamp or sticker  230 . Six separate assays are to be performed using a six well tray. Each well  24  will have a reagent A′-F′ which reacts, respectively, with A-F. Control A should be positive only on the slide  70  placed onto well  24  with reagent A′ and should be negative for the remaining 5 wells. Control B should be positive only on slide  70  placed onto the well  24  with reagent B′ and should be negative for the other 5 wells, etc. The stamps or stickers  230  with these external controls can be premade commercially for mass sale or they can be custom made. It is also useful if a stamp or sticker  230  for a common clinical panel of assays is color coded or otherwise labeled so that a quick glance is indicative of the assays being performed. This color code or other labeling can also be matched to the color code or other labeling of trays  14  to be used in conjunction with the stamp, e.g., a green stamp will have antigenic determinants A-F on it and a green tray will have antibodies A′-F′. A numbering or lettering system can be used as one alternative to a color coding scheme. These could be used for a series of tests for breast cancer whereas a red stamp and red tray could indicate those to be used to assay for Hodgkin&#39;s disease. Any type of color coding, such as a series of stripes of colors, can be used. Such color coding will result in fewer errors being made in the clinical laboratory. The use of the positive control on each slide also acts as an automatic labeling system for the slide since the positive external control is indicative of the assay performed for that slide. If desired, the stamps can be packaged with their corresponding trays and can even be placed onto each tray when packaged and then peeled from the tray and placed onto a slide at the time of use. The use of such stamps or stickers with controls on them is much simpler and less time consuming than preparing a control biological sample, e.g., a tissue section of normal or neoplastic tissue, to be used as such a control. 
     As an example, a breast panel of assays can be performed in which six distinct diagnostic markers are used. These diagnostic markers can be cytokeratin 7, cytokeratin 20, ER, Bcl-2, PR, and cathepsin D. Each of these antigenic determinants can be coated onto a stamp or sticker to be used as controls and the corresponding antibodies can be predried on separate wells of a 6 well tray. If cytokeratin 7 or an equivalent antigenic determinant is placed on position A of the stamp or sticker, then antibody against cytokeratin 7 is to be placed in well A′. Section A of the stamp or sticker should be positive on the slide placed on well A′ but should be negative on the other 5 wells. Also, only section A of the stamp should be positive on the slide  70  placed on well A′, while sections B-F of the stamp or sticker should be negative. This results in the automatic labeling of the slide by the built-in control. If section A is not positive or if any of sections B-F are positive on this slide it means that a problem has occurred and the test should not be relied upon. 
     Other examples of panels which may be used are a panel of prognostic markers for breast cancer such as Ki-67, Her-2/neu (c-erbB-2), P53, pS 2 , EGFR, and Factor VIII. Other neoplasms, e.g., prostate, bladder and colon can also use the same prognostic panel tray. In general pathology practice, four panel trays can cover 90-95% of diagnoses of all hemopoietic diseases: 1) A Hodgkin&#39;s disease panel may include the markers LCA (CD45), L26 (CD20), CD3, Leu-M1 (CD15), Ki-1 (CD30), and LMP. 2) A non-Hodgkin&#39;s panel can include L26 (CD20), CD3, MT1, Bcl-1, Bcl-2, Ki-1 (CD30). 3) A separate non-Hodgkin&#39;s panel can include Kappa, Lambda, UCHL-1 (CD45RO), CD5, CD23, and CD10. 4) A leukemia panel can include L26 (CD20), CD34, MPO, Lyso, TdT. and DBA44. Any other desired panel of tests can be similarly performed, such as but not limited to, panels for undifferentiated tumor of unknown primary site, sarcoma classification, lymphoma vs. carcinoma vs. melanoma, adenocarcinoma vs. mesothelioma, hepatocellular/cholangiocarcinoma vs. metastatic carcinoma, pituitary panel, Paget&#39;s disease vs. melanoma vs. squamous cell carcinoma vs. fibrous histiocytoma, breast panel, and bladder vs. prostate carcinoma. Yet other possible panels are a neuroendocrine panel, small round cell tumor, germ cell tumor, Hodgkin&#39;s vs. non-Hodgkin&#39;s lymphoma, lymphoma vs. reactive hyperplasia, plasma cell dyscrasia, leukemia panel and a virus panel. 
     Each laboratory can devise its own system which is most appropriate to the personnel and to the number and types of assays being performed. For example, if an assay requires use of a first set of antibodies followed by reaction with a secondary antibody wherein the secondary antibody is identical for all samples, then if a small number of assays are to be performed one may do these on the trays  14 , but if a large number of assays are being performed one may prefer to place all the slides into a large tank with the secondary antibody and/or detection system (a “batch” or “bulk” incubation method. Alternatively, for the lab doing a small number of assays, it is possible to coat a piece of filter paper with the secondary antibody and/or detection system, lay all the slides onto the filter papers and wet the filter paper at the time of use. This can be less expensive than using the trays. Similarly, nucleic acid probes can be placed onto the filter paper. 
     Example 7 
     Built-in Controls—Nucleic Acid Hybridization 
     In a manner similar to that discussed in Example 6 for immunoassays, built-in controls can be used for nucleic acid assays such as ISH or fluorescent in situ hybridization (FISH). In one type of FISH, fluorescent probes are used which illuminate large portions of the chromosomes. This is referred to as whole chromosome painting (WCP). This technique is useful for observing gross chromosomal aberrations such as translocations. The probes used can be in conjunction with a variety of different colored fluorophores. For example, probes to chromosome 1 can fluoresce orange, probes to chromosome 2 can be made to fluoresce green and probes to chromosome 3 can use a red fluorescing fluorophore. It is therefore possible to stain for all three chromosomes simultaneously and still be able to easily distinguish them from each other. In human cells, there can be up to 24 distinct nuclear chromosomes, these being chromosomes 1-22, X and Y. If three different fluorophores are used, all 24 chromosomes can be studied by using only 8 different tissue sections or 8 different sets of cells. These can be studied on 8 separate slides or if desired several tissue sections or sets of cells can be placed on separate sections of a single slide. It is possible to place 8 tissue sections on a single slide and thereby study all 24 chromosomes on a single slide with all reactions being performed simultaneously using 8 different sets of three mixed probes. These can be tested on a single cell smear slide by placing the slide on a tray or chip with 8 separate wells wherein each well has had predried in it a different set of 3 probes. Using microarray techniques, 24 built-in controls will be directly coated on the slide such that they will surround, within the inner borders, each well region (see FIG.  16 E). One of skill in the art recognizes that it is not necessary to use 8 sets of 3 probes. Other variations are possible such as 6 sets of 4 differently labeled probes. It is also not necessary to use trays with predried reagents, rather the reagents can be added to the trays in liquid form. In a similar fashion, other techniques, such as in situ hybridization, can be performed using a desired number of controls which have been directly coated onto the slide in the region surrounding the inner borders of the wells. Although the controls have been shown as placed on the slide so as to surround the edges of the wells, such a pattern is not required and other patterns of arranging the controls can be used so long as they are in a region which contacts the reagents in the wells. 
     Example 8 
     Automated Multiwell Tray and Machine 
     Analysis of biological samples is very labor intensive, even with the use of automated systems since the automated systems still require several steps to be performed manually. A multiwell tray, or a multiwell tray with predried reagents, attached to tubing and a pump or pumps or connected to an automated processing machine can be used to partially or completely automate the processing of biological samples. Such a multiwell tray can be similar in design to the tray  14  discussed earlier. But the automated multiwell tray  330  (see FIGS. 15A-B) is used for steps such as washing or with less expensive reagents which can be used in larger amounts. The reaction chamber  280  of the automated multiwell tray  330  is designed to hold volumes such as 0.01-1 mL, although this amount is not critical and can be larger or smaller. The well includes one or more inlets and one or more outlets to accommodate tubing. The tubing entering an inlet is attached to a pump. A slideholder  1  with attached slides  70  is placed on top of the automated multiwell tray  330  and fluids can be pumped into the reaction chambers  280  through an inlet such as  300  or  302 . Reagents can be recirculated during the reaction time and reused if desired (e.g., as shown in FIG. 15B) by using a pump  290  and tubing  295  through inlet  302  in conjunction with tubing  310  through outlet  294 . Alternatively one can send the used material directly to a waste container  291  or a sink or to be analyzed, such as on a gel or by other instrumentation, via outlet  296 . Circulated reagents can reduce incubation or reaction time and reduce background. The concentration of circulated reagents also can be gradually increased or decreased to reach the optimal reactive condition, especially when using multiple probes. This is especially applicable when a soft bottom tray is used which allows the use of varied volumes. 
     A central processing unit  286  controls the pumping of reagents and can open and close valves on various pieces of tubing attached to a pump so that one pump can control several different reagents or alternatively multiple pumps can be used all controlled by the central processing unit. With this setup, a slideholder with slides and mounted biological samples can be placed onto a multiwell tray, the central processing unit can be activated to pump desired fluids and reagents into the reaction chambers either recirculating the fluids or disposing of the fluids directly. Different reagents can be pumped into the reaction chamber sequentially without the need of a person transferring the slides from one tray to another tray. For example, slides with biological samples can be placed onto the automated multiwell tray and the system can pump in the reagents: xylene, 100% ethanol, 90% ethanol, hydrogen peroxide, a secondary antibody, detection reagents (ABC), diaminobenzidine, hematoxylin, PBS wash solution between each step, and the further 90% ethanol, 100% ethanol and xylene and a coverslipping solution. The slides can be removed from the automated multiwell tray for any desired intervening steps for which it is desirable to have the reaction performed on a regular multiwell tray  14  as described earlier. 
     As another example, slides with a mounted tissue section can be deparaffinized and treated separately and then placed onto a multiwell tray which has predried reagents and then be attached to the automatic processing machine which will pump in the desired reagents, e.g., secondary antibody, detection reagents (ABC), diaminobenzidine and hematoxylin as well as PBS wash buffer between each of these steps, followed by 90% ethanol, 100% ethanol, xylene and a coverslip solution. 
     The use of the automated multiwell tray has several advantages. It allows several steps to be done in succession with no manual labor required at each step. It also is safer because some dangerous chemicals, e.g., xylene and diaminobenzidine which are carcinogens, can be pumped directly from a container into the reaction chamber and from there into a waste receptacle or a receptacle from which the reagents can be reused without the need of a person pipetting these reagents into wells and handling the trays with these carcinogens on them. Recycling of such reagents using the prior art method of simply dropping reagents on top of biological samples mounted on slides is impracticable. Therefore the automated multiwell tray reduces exposure to hazardous chemicals, makes it easy to dispose of hazardous chemicals, and also reduces use of such chemicals because they can be reused and recycled. 
     The central processing unit  286  can also control heating and cooling of a heat block  288  to perform automated in situ PCR or to denature a probe being used for in situ hybridization. PCR reagents, including biotin or digoxigenin if desired, and primer sets can be coated and dried onto the wells of the tray  330 . The slide  70  with sample  220  is placed onto the tray  330  and water or buffer is added. The heating block  288  can be placed against the slide  70  (as shown in FIG. 15B) or the tray  330  or can be one designed to contact both sides of the slide plus tray assembly and can be controlled by the central processing unit  286 . Two results can be obtained from each well  410 . First, fluid from a well  410  can be removed and assayed on a gel  298  to determine whether a band of DNA is seen. The size of any such band can also be determined on the gel  298 . This acts as a control to see whether the PCR has worked successfully. This is possible because a large fraction of the amplified DNA does not remain in the cells of the sample but leaks out to the fluid in the well. Second, a fraction of the amplified DNA remains in the cells and this can be observed by detecting the biotin or digoxigenin by methods well known to those of skill in the art. Thus an in situ PCR shows which cells are detected by the assay. 
     The present invention also uses a novel modification which allows one to recover the reaction fluid and to assay this fluid, prior to continuing the work-up of the tissue sample, to determine whether the PCR has worked properly or has been contaminated. This assay is extremely quick and simple, e.g., simply running the reaction fluid on an agarose gel and looking for the presence of a specific band size. In the event that one determines that the PCR did work properly, then it is worth continuing the workup of the tissue sample. However, if it is determined that the PCR failed, one knows that it is not worth the labor and expense of continuing with the particular sample. 
     The above noted ability to assay the reaction fluid is useful not only for determining whether it is worth continuing to workup the specific sample, but this ability also yields data not available from viewing only the in situ hybridization results within the tissue. When in situ hybridization is performed, some fraction of amplicons remains where it was amplified while the rest ends up in the solution. By assaying the portion in solution, one can determine not only a relative amount of nucleic acid, but one is also able to determine the size of the amplified nucleic acids. When one views only the tissue sample one cannot determine the size product which is formed, one learns only that some nucleic acid was amplified and one also learns which cells were expressing the nucleic acid. These two sets of data are complementary. It is apparent that the present invention allows one to view both sets of results with the data of both being complementary. To date no apparatus has been available which had allowed one to obtain both types of data from a single polymerase chain reaction. 
     A further aspect of the invention is that the volume of the reaction chamber  280  is adjustable. Preferably a central processing unit  286  controls a piston  284  which pushes against reaction chamber bottom  282  which is either flexible or movable. This movement adjusts the volume of space in the reaction chamber  280 . For example, when performing in situ PCR, it is desirable to keep the reaction volume very small, e.g., 10-50 μL. Following the PCR reaction it may be desired to pump the reaction fluid out of the reaction chamber. However, such a small volume of fluid will be held between the slide  70  and reaction chamber bottom  282  by capillary action. By allowing the reaction chamber to be enlarged to encompass more fluid, it becomes easier to accomplish the desired pumping. Those of skill in the art recognize that a variety of means can be used to adjust the volume of the reaction chamber  280 . It is not necessary to use a piston controlled by a central processing unit. For example a screw means can be placed against the reaction chamber bottom and by turning the screw means the screw means will press against the tray bottom to force the bottom of the reaction chamber toward the microscope slide to reduce the volume of the reaction chamber  280 . Reversal of this process again enlarges the volume. 
     Example 9 
     Whole Chromosome Painting 
     Chromosomes can be examined for gross abnormalities such as translocations by a technique known as whole chromosome painting. This method uses a number of fluorescently labeled probes which bind to a chromosome effectively to “light up” the whole chromosome. Sets of probes specific for each chromosome can be used to study any desired chromosome. Humans have a total of 24 nuclear chromosomes, these being chromosomes 1-22, X and Y. It is common to paint multiple chromosomes at one time. The chromosomes are easily distinguished by using fluorescent probes of different colors. For example, chromosomes 1, 2 and 3 can be stained simultaneously by using probes which fluoresce orange for one chromosome, probes which fluoresce green for a second chromosome, and probes which fluoresce red for a third chromosome. Using such a system, one test would typically use 8 slides of cells to examine the complete nuclear genome of a human. This test would include the placing the 8 slides onto 8 wells of a tray. One example of tissue to be assayed is a blood or bone marrow smear. The probes can be predried in the wells if desired. 
     A chip or tray  400  designed to allow the analysis of all 24 chromosomes on a single slide  70  is presented here. The tray  400  is one which can snap on to or otherwise be attached to a microscope slide  70 . The chip or tray  400  contains 8 wells  410  with each well  410  separated from neighboring wells  410  by a gap or a trough  420 . Such a tray  400  is illustrated in FIG.  16 A. Each well  410  in the tray  400  has a narrow opening  430  through which reagents can be added to the wells  410 . 
     In practice, cells to be examined are dropped or spread across a microscope slide  70 . The slide  70  is then attached to the tray  400  such that the cells are facing the wells  410  of the tray  400 . Reagents are then added to each well  410  individually through the opening  430  in the tray to each well  410 . The reagents will spread between the well  410  and the slide  70  by capillary action. Different reagents specific for the various chromosomes are added to each well  410 . The gap or trough  420  between wells  410  prevents the reagents from one well  410  spreading to a neighboring well  410  thereby preventing cross-contamination. The wells  410  hold a predetermined amount of fluid, e.g., 10-20 μL each, and capillary action allows only enough buffer to be added to fill the wells  410  without causing excess overflow. This aids in preventing cross-contamination. Three different chromosomes can be assayed in each well  410  using, e.g., orange, green and red fluorescent probes thereby allowing all 24 human nuclear chromosomes to be assayed on a single slide  70 . 
     In a preferred embodiment, the probes are predried onto the 8 wells  410  of the tray  400  with probes for 3 different chromosomes in each well  410 . If desired, other reagents such as salts can also be predried into each well  410 . Metaphase or interphase cells are fixed across a slide  70  and the slide  70  is placed in contact with the tray  400 . Then buffer is added to the openings  430  to each well  410 . With this method, there is no necessity to pipet the different reagents into each well  410 , rather the same buffer is added to all wells  410  thereby preventing the possibility of pipetting incorrect reagents (human error) into wells  410 . The predried probes and salts dissolve upon addition of buffer to the wells  410  and hybridization is allowed to occur. A typical incubation may be at 70-90° C. for 1-2 minutes to denature the probes as well as the cellular DNA followed by an incubation at 37-45° C. for approximately 2 hours, although it is common to perform incubations for anywhere from 30 minutes to overnight. The hybridization buffer can be chosen as desired with several buffer systems commonly used in the art. For example 2×SSC is commonly used. Formamide is sometimes added to the buffer. In a preferred embodiment, following incubation the tray  400  can be placed onto a blotting material, e.g., paper towels, and the reaction fluid in the wells  410  will be physically removed from the wells  410  by capillary action, the blotting material soaking up the hybridization fluid. This prevents cross-contamination between wells  410  when the slide  70  is separated from the tray  400 . 
     In a more preferred embodiment, the slide  70  includes positive and negative controls in the regions  440  which are those which are in contact with the hybridization fluid in each of the 8 wells  410 . Using microarray technology which has become quite popular recently, nucleic acids which are complementary to the probes being used to paint the chromosomes are coated and immobilized onto the slide  70 , preferably prior to placing cells upon the slides  70 . This may best be performed under industrial conditions and the slides  70  can be sold with the controls built in. It is preferred that 24 controls  442  are placed onto each slide  70  at all 8 regions which are to be in contact with hybridization buffer. One example of an array is shown in FIG. 16E in which all 24 nucleic acids are arrayed around the edges of each region  440  which will contact each of the 8 wells  410 . If for example, a first region  440  is one which will contact a well  410  containing probes for chromosomes 1, 2 and 3, then the control nucleic acids for these chromosomes should light up after staining (each showing only a single color) while the remaining 21 controls should not hybridize and should not fluoresce. In this manner there are both positive and negative controls and labels for each of the 8 wells  410 . 
     One of skill in the art recognizes that other similarly designed trays can be utilized. There is no need for an 8 well tray. For example, if 4 differently colored fluorescent probes are to be used, the same results could be obtained with a 6 well tray. Furthermore, this invention is not limited to the analysis of human chromosomes. Chromosomes from any other organism can be similarly examined and the number of wells on the tray is a matter of personal choice, often determined by the number of chromosomes or probes to be examined. One of skill in the art also recognizes that trays can be designed to hold more than a single slide such that multiple cell samples can be assayed at once, with the multiple slides being handled together more easily than several separate slides. 
     Use of the above methods allows one to obtain results of a whole panel of markers in as little as 15-30 minutes. Thus the results can be obtained while the patient is still in the operating room. The pathologist and surgeon can decide immediately whether to perform more surgery or if chemotherapy or radiation treatment is necessary. This can allow the surgeon to proceed immediately rather than having to perform more surgery at a later date. If the currently sold automated system were used instead of the methods of the instant invention, it would take longer to receive results, partially because the currently sold automated system does not assay one patient at a time but rather many samples are loaded into the automated instrument at one time and it is necessary to wait while they are all loaded and then processed. The currently sold automated system drops reagents on top of slides and the biological sample is not always completely covered, whereas the present method of placing a biological sample on top of a well filled with reagents ensures that the whole sample is in contact with reagent. 
     The above Examples are only exemplary and not meant to be limiting of the techniques which may be performed using the apparatus which is defined by the present invention. The invention is applicable to, but not limited to, immunohistochemistry, in situ hybridization, in situ PCR, and fluorescent in situ hybridization (FISH). The stated measurements are also exemplary and not meant to be limiting as it will be obvious to one of skill in the art that the exact measurements are not critical and can be varied to still yield successful results. Those skilled in the art will readily perceive other applications for the present invention. 
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