Abstract:
Apparatus and method for Immunohistochemistry, including a slide holder assembly fixture, a slide holder, a slide holder frame, and a slide holder frame assembly adapted to be placed in communication with a driving force to draw fluid through the slide holder. The slide holder may include an injection port that allows consistent fluid delivery and recovery, while minimizing the introduction of air bubbles. The slide holder may include a reservoir that allows for filling, soaking, rinsing, flushing, uniform antibody coverage of the tissue and reduces handling time, space, and errors, and one or more sample chambers. The slide orientation in the slide holder allows for visualization of label information and tissue to confirm adequate antibody coverage. A multi-port frame allows multiple assays to be carried out concurrently, and is removable from a vacuum manifold and allows for easy transport from the vacuum manifold to an incubator/refrigerator and back.

Description:
[0001]    This application claims priority of U.S. Provisional Application Ser. No: 61/951,135 filed Mar. 11, 2014 and 62/039,082 filed Aug. 19, 2014, the disclosures of which are incorporated herein by reference. 
     
    
     BACKGROUND 
       [0002]    Immunohistochemistry (IHC) refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting specific antibody-antigen interactions using labeled antibodies or other ligands. IHC is also widely used in basic research to understand the distribution and localization of biomarkers and differentially expressed proteins in different parts of a biological tissue. Applications include diagnostics, pharmaceutical development and research. 
         [0003]    For example, samples can be prepared on individual slides, or multiple samples can be arranged on a single slide for comparative analysis, such as with tissue microarrays. IHC slides can be processed and stained, and then viewed by either light or fluorescence microscopy. The main output of the tissue staining is for optical or fluorescence imaging as well as archival and storage of stained tissue. 
         [0004]    Currently there are high and moderate volume automated systems available for IHC. Manual systems are available for the occasional research, which are labor intensive and require many pipetting and dip/soak washing steps. These steps include slide mounted tissue washing, blocking, primary, secondary and tertiary antibody introduction, covered incubation, as well multiple washes and rinses in between each step. This complex, lengthy handling process is prone to errors, which can ultimately compromise the result. 
         [0005]    It would be desirable to provide a system that eliminates drawbacks of the conventional systems, allowing, for example, consistent fluid delivery to the sample, recovery of precious reagents, minimum slide handling (no transfer of slides between different baths) and carrying out of multiple concurrent assays. 
       SUMMARY 
       [0006]    Drawbacks of the prior art have been overcome by the embodiments disclosed herein, which in certain aspects relate to a vacuum source connected to a flow cell sandwiched onto a sample mounted slide. The cell keeps the tissue from drying out, and allows fluids (e.g., blocking agents, antibodies, wash and rinses, etc.) to be introduced and placed into contact with the sample as well as flushed out of the chamber. In certain embodiments, the apparatus includes a slide holder assembly fixture, a slide holder, a slide holder frame, and a slide holder frame assembly adapted to be placed in communication with a driving force such as vacuum to draw fluid through the slide holder. 
         [0007]    In certain embodiments, the slide holder includes an injection/recovery port that allows consistent fluid (e.g., antibody, linker, reporter and chromogen) delivery and recovery, while minimizing the introduction of air bubbles, thus reducing error and allowing about &gt;75% fluid (e.g., antibody) recovery, and in certain embodiments, &gt;95% recovery. In certain embodiments, the slide holder includes a reservoir that allows for filling, soaking, rinsing, flushing, uniform antibody coverage of the tissue and reduces handling time, space, and errors. In certain embodiments, the slide orientation in the slide holder allows for visualization of label information and tissue to confirm adequate antibody coverage. The multi-port frame allows multiple assays to be carried out concurrently (e.g., 1-12 slides can be processed per frame assembly). The slide holder frame is removable from a vacuum manifold and allows for easy transport from the vacuum manifold to an incubator/refrigerator and back. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0008]      FIG. 1  is an exploded view, in perspective, of a slide holder assembly fixture and slide in accordance with certain embodiments; 
           [0009]      FIG. 2  is an exploded view, in perspective, of a slide holder assembly fixture with a mounted slide, and a slide holder, in accordance with certain embodiments; 
           [0010]      FIG. 3  is a perspective top view of a slide holder mounted on a slide holder assembly fixture, in accordance with certain embodiments; 
           [0011]      FIG. 4  is a perspective view side view of a slide holder mounted on a slide holder assembly fixture, in accordance with certain embodiments; 
           [0012]      FIG. 5A  is a front view of a slide holder in accordance with certain embodiments; 
           [0013]      FIG. 5B  is a side view of the slide holder of  FIG. 5A  in accordance with certain embodiments; 
           [0014]      FIG. 5C  is an enlarged perspective view of a portion of the gasket in the slide holder in accordance with certain embodiments; 
           [0015]      FIG. 6  is a first perspective view of a portion of the slide holder of  FIG. 5A  in accordance with certain embodiments; 
           [0016]      FIG. 7  is a second perspective view of a portion of the slide holder of  FIG. 5A  in accordance with certain embodiments; 
           [0017]      FIG. 8  is a front view of the slide holder of  FIG. 5  showing the direction of fluid flow into the slide holder chamber in accordance with certain embodiments; 
           [0018]      FIG. 9  is a front view of the slide holder of  FIG. 5  showing the direction of fluid flow out of the slide holder chamber in accordance with certain embodiments; 
           [0019]      FIG. 10  is a perspective view of a slide holder frame and cover holding slide holders in accordance with certain embodiments; 
           [0020]      FIG. 11  is a cross-sectional view of a slide holder positioned in a slide holder frame in accordance with certain embodiments; 
           [0021]      FIG. 12  is a perspective view of a slide holder frame in accordance with certain embodiments; 
           [0022]      FIG. 13  is a perspective view of a slide holder and slide holder assembly fixture showing a slide release feature in accordance with certain embodiments; 
           [0023]      FIG. 14  is a perspective view of a slide having a membrane attached thereto in accordance with certain embodiments; 
           [0024]      FIG. 15A  is an exploded perspective view of the slide of  FIG. 14  and a vacuum manifold having slide wells, in accordance with certain embodiments; 
           [0025]      FIG. 15B  is a perspective view, in cross-section, of slide wells of a vacuum manifold, in accordance with certain embodiments; 
           [0026]      FIG. 15C  is an exploded cross-sectional view of a slide and slide wells of a vacuum manifold, in accordance with certain embodiments; 
           [0027]      FIG. 16A  is a front view of a slide holder in accordance with certain embodiments; 
           [0028]      FIG. 16B  is a first perspective view, in cross-section, of a slide holder in accordance with certain embodiments; 
           [0029]      FIG. 16C  is a second perspective view, in cross-section, of a slide holder in accordance with certain embodiments; 
           [0030]      FIG. 16D  is top view of a slide holder in accordance with certain embodiments; 
           [0031]      FIG. 17A  is an exploded perspective view of a slide and slide holder flow cell assembly in accordance with certain embodiments; 
           [0032]      FIG. 17B  is a perspective view of the slide mounted to a slide holder flow assembly in accordance with certain embodiments; 
           [0033]      FIG. 17C  is a bottom perspective view of the assembly of  FIG. 17B ; 
           [0034]      FIG. 18A  is an exploded perspective view, in cross-section, of a manifold frame and slide holder flow cell in accordance with certain embodiments; 
           [0035]      FIG. 18B  is a perspective view, in cross-section, of the manifold frame and slide holder flow cell of  FIG. 18A  with the flow cell in place, in accordance with certain embodiments; 
           [0036]      FIG. 18C  is a side view, in cross-section, of the assembly of  FIG. 18B ; 
           [0037]      FIG. 19  is an exploded view of a slide holder flow cell and slide in accordance with certain embodiments; 
           [0038]      FIG. 20A  is a perspective exploded view of a flow cell and slide in accordance with another embodiment; 
           [0039]      FIG. 20B  is a perspective view of the flow cell of  FIG. 20A  with a slide in place, in accordance with certain embodiments; 
           [0040]      FIG. 20C  is a perspective view of the flow cell of  FIG. 20A  in an assembled condition, in accordance with certain embodiments; 
           [0041]      FIG. 21  is a schematic diagram of the flow cell of  FIG. 20A  shown positioned in a vacuum manifold in accordance with certain embodiments; and 
           [0042]      FIG. 22  is a schematic view of the lower region of the flow cell of  FIG. 20A  shown positioned in a vacuum manifold in accordance with certain embodiments. 
       
    
    
     DETAILED DESCRIPTION 
       [0043]    Turning first to  FIG. 1 , there is shown a slide  10  having a sample  11  thereon, such as a tissue sample. Typically the tissue sample has already gone through a fixative process before it is placed on the slide  10 . The slide  10  is a conventional sample holder and can be planar and made of glass, for example. Also shown is sample or slide holder assembly fixture  20 , which receives slide  10  in mounting engagement. In certain embodiments, the sample  11  is oriented on the topside of the slide  10 , and when placed into position, resides within a predetermined sample target region (arrow A) of the slide holder assembly fixture  20 . In certain embodiments, one or more level indicators  12  (three shown) are positioned in the sample target region, which assist the operator in positioning of sample in a manner that ensures sample coverage even in a half-filled sample chamber, thus using a minimal amount of primary antibody. 
         [0044]    In certain embodiments, the slide holder assembly fixture  20  is an elongated member having a generally flat top surface  21 , an end wall  22 , and an opposite end wall  23 . In certain embodiments, the end wall  22  extends vertically above the surface  21  a higher distance than the end wall  23 . The length between the inwardly facing surfaces of the end walls  22  and  23  is slightly longer than the length of slide  10 , so that slide  10  fits between the end walls  22 ,  23  as shown in  FIG. 2 . Short side walls  24 ,  25 , as well as alignment fins  26 ,  27 , are arranged on the perimeter of the surface  21  and extend vertically upwardly from the surface  21 , and also serve to contain the slide in position on the surface  21  of the slide holder assembly fixture  20 . Alignment fins  26 ,  27  also serve as alignment members for positioning the slide holder  30 , as discussed in greater detail below. In certain embodiments, spaced cut outs  28  (four shown) are formed in the surface  21  of the fixture  20 . In certain embodiments, each cut out  28  is shaped as a partial truncated cone, with its inwardly deepest cut portion positioned at the surface  21 , tapering outwardly towards the side perimeter as the cut approaches the bottom of the fixture  20 . The cut outs  28  are configured to receive respective compression clips  43  of slide holder  30 , as discussed in greater detail below. 
         [0045]    In certain embodiments, slide holder  30  is a single use device and includes a reservoir  31  and a sample chamber  33  in fluid communication with the reservoir  31  via narrow slit  32  at the bottom of the reservoir  31  that communicates with a reservoir channel  34  opening into the sample chamber  33 . In certain embodiments, the reservoir  31  is configured to hold up to 20 ml of fluid volume. In certain embodiments, the slide holder  30  may be made of polycarbonate, polystyrene, polyethylene or derivatives (e.g., PETG, PET, PE, PETE), polypropylene or acrylic. In certain embodiments, the sample chamber  33  includes a gasket  35 , such as a dispensed silicon gasket, as seen in  FIG. 5A . In certain embodiments, the gasket  35  also could be overmolded, such as overmolded thermoplastic elastomer (TPE), overmolded silicone, diecut silicone, diecut double stick foam, thick diecut acrylic adhesive, etc. The gasket  35  includes a region that delimits an injection/recovery port fluid channel  36 . The injection/recovery port fluid channel  36  is in fluid communication with an injection/recovery port channel port  37 , that in turn is in fluid communication with injection/recovery port  38  configured to receive an injecting device such as a pipette  39 . The injection/recovery port fluid channel  36  opens into sample chamber  33  at outlet opening  40 , best seen in  FIG. 5C . 
         [0046]    In certain embodiments, slide holder  30  includes a drain port  41  that drains fluid from the chamber  33  to drain  42 . In certain embodiments, slide holder  30  includes spaced slide compression clips  43 , each of which is received by and engaged in a respective cut out  28  of the slide holder assembly fixture  20 . In certain embodiments, the compression clips  43  terminate in a free end  44  and include ramp notches  45  ( FIG. 4 ) that provide flexibility to the compression clips  43 . 
         [0047]    In certain embodiments, to attach the slide  10  to the slide holder  30 , the slide holder  30  is positioned above the slide as shown in  FIG. 2 , and is pressed down onto the slide holder assembly fixture  20 , aligning with the alignment fins  26 ,  27 , the side walls  24 ,  25  and the end wall  22  ( FIG. 3 ). The ramp notches  45  that deflect the slide compression clips  43  allow for optimum engagement in the cut outs  28 . In this assembled state, the sample  11  supported on the slide  10  is positioned in the sample chamber  33  of the slide holder  30 . Suitable fluids can be introduced into the sample chamber  33 , such as via pipette  39  or by the reservoir  31 . For example, the tip of pipette  39  can be inserted into the injection/recovery port  38  as best seen in  FIGS. 6 and 7 . Fluid from the pipette  39  flows into the injection/recovery port fluid channel  36 , outlet opening  40  and into sample chamber  33  where it contacts the sample. 
         [0048]    Turning now to  FIG. 8 , the arrows depict the flow of fluid, such as antibodies, linkers, reporters, and chromogens which can be introduced into the injection/recovery port  38  via pipette  39 , for example. The fluid travels down the injection/recovery port fluid channel  36 , out outlet opening  40 , and into the sample chamber  33  where it fills the chamber from the bottom up, thus reducing potential bubbles that could cause inconsistent coverage of the sample supported on the slide  10 . 
         [0049]      FIG. 9  depicts the flow of fluid during removal or recovery of the fluid from the sample chamber. Here, the injection/recovery port  38  is used as a retrieval port by using the pipette  39  to suck out fluid from the sample chamber to recover &gt;75% of the fluid. In certain embodiments, as much as 95% recovery is achieved. High recovery can be important, particularly where the fluid comprises precious or custom antibodies. 
         [0050]    Turning now to  FIGS. 10 and 11 , as assembly  200  is shown, which includes manifold base  100 , sample or slide holder frame  102 , and cover  101 . In certain embodiments, the manifold base  100  is in communication with a driving force (not shown), such as a vacuum, to drive fluid through one or more slide holders  30  mounted on a slide holder frame  102  positioned on the manifold base  100 , which can serve as a wash basin for multiple rinses, washes, etc. of the sample. In certain embodiments, one or more covers  101  can be positioned over the one or more slide holders  30  to prevent light from affecting any fluorescent signal chemistries involved in the assay and eliminating potential contamination of samples. In certain embodiments, the manifold base  100  can accommodate one or more slide holder frames  102 . Each slide holder frame ( 102 ) can hold, for example, 1-12 slide holders  30  in communication with the driving force. The slide holder frame  102  can be removable from the manifold base  100 . 
         [0051]    In certain embodiments, cover  101  can include a pinch handle  112  to assist in lifting cover  101  off the slide holder frame  102 . In certain embodiments, the cover  101  can include a task tracker  103  to track which step in the protocol is in process, which is useful when multiple slide holder frames  102  are being used simultaneously, or are residing in an incubation chamber or refrigerator. In certain embodiments, the task tracker  103  is a movable dial (with respect to the cover surface) with one or more stationary markings that point to other markings on the cover  101 . In certain embodiments, the cover  101  may also have a plurality of spaced stacking indents  104  each configured to receive a respective leg  114  ( FIG. 11 ) of another slide holder frame  102  to enable stacking for economy of space such as may be necessary when placed in an incubation chamber. 
         [0052]    In certain embodiments, the slide holder frame  102  includes a plurality of ports  105  ( FIG. 12 ), each configured to receive a respective slide holder  30 . The ports can be numbered as shown for facilitating the tracking of each individual assay. In the embodiment shown, each slide holder frame  102  is configured to hold up to  12  slide holders  30  along with  12  port plugs  106 , although those skilled in the art will appreciate that fewer or more could be used. If a port  105  is not occupied by a slide holder  30 , a port plug  106  can be installed to retain vacuum in the system. When ports  105  are in use (e.g., are occupied by a slide holder  30 ), the plug  106  for that port  105  can be stored in a convenient storage aperture  148  next to the installed slide holder  30 . In certain embodiments, the slide holder frame  102  can include a well  107  for storage of the slide holder assembly fixture  20 . Activation of the driving force such as vacuum allows for simultaneous fluid flushing of multiple slide holders  30  positioned in the slide holder frame  102 . 
         [0053]      FIG. 11  illustrates certain embodiments of the slide holder  30  positioned in the slide holder frame  102 . In accordance with certain embodiments, spaced tabs  48  on the bottom of the slide holder  30  are received by corresponding slots  149  in the slide holder frame  102 . When slide holder  30  is so positioned in the slide holder frame  102 , the drain  42  penetrates into port  105  and is sealed by port seal  108 . A valve  110 , such as a duckbill valve, is positioned in the port  105  and prevents fluid from exiting the slide holder chamber  33  until the valve is overcome by the application of vacuum to the port  105 . 
         [0054]    In certain embodiments, blocking, rinsing and washing steps are achieved by loading up the reservoir  31  such as with a repeating pipette, squirt bottle or any dispensing device. A solution is loaded and flows into the sample chamber  33  and is allowed to soak the sample. The vacuum is activated and the solution flows over the sample and out the drain  42 , rinsing off any residual blocker, antibodies, linkers, reporters, chromogens or hemotoxilins, for example. 
         [0055]    In certain embodiments, slide  10  supporting tissue sample  11  is placed in the slide holder assembly fixture  20  with the tissue side facing upwardly. The slide  10  is placed to ensure that the region containing the tissue is within the target area so as to not interfere with the gasket seal of the slide holder  30 , as aided by indicators  12 . The slide holder  30  is then aligned onto the slide holder assembly fixture  20  and pressed firmly onto the slide  10  until the compression clips  43  engage in the respective cut-outs  28 . 
         [0056]    This assembly is next plugged into a desired port inserted into the slide holder frame  102  on the base  100 . The sample port position can be recorded if needed. Any unused ports in the frame  102  are plugged with a port plug  106  as necessary. In certain embodiments, the plane of the slide is thus perpendicular to the plane of the surface of the slide holder frame  102 ; it is positioned in a horizontal/vertical plane to allow view of sample and slide information, and minimize fluid chamber volume. 
         [0057]    If hydration or washing is necessary, the desired solution is introduced into the reservoir  31  such as to hydrate or wash the tissue sample, for a desired time period. Vacuum can then be applied to the base to wash and flush the solution from the slide holder. 
         [0058]    Blocking can be carried out by loading the desired concentration of a blocking agent into the injection/recovery port  38  or the reservoir  31  of the sample chamber  33 . Enough blocking solutions should be loaded to cover the sample  11  in the sample chamber  33 . 
         [0059]    Once all of the slide holders are filled, and if incubation is needed, the slide holder frame is covered with cover  101 , and the task tracker  103  can be set to the appropriate task, such as the “blocker” indication. The covered frame  102  may then be incubated, such as at room temperature, in an incubator, or in a refrigerator for the desired time. 
         [0060]    After incubation, the blocking agent can be flushed by returning the frame  102  to the base  100 , removing the cover, and applying vacuum, and any necessary wash steps carried out by introducing wash solution to the reservoir  31 , allowing the sample  11  to soak (if needed) in the wash solution for the desired time, and then flushing by applying vacuum. A plurality of such wash steps can be employed if desired. 
         [0061]    Primary antibody of the desired concentration can be introduced into the injection/recovery port  38  in an amount sufficient to cover the sample  11 . If an incubation step is desired, the frame  102  is covered with cover  101 , and the task tracker set to the appropriate indication, such as “primary”. The covered frame  102  may then be incubated, such as at room temperature, in an incubator, or in a refrigerator, for the desired time. 
         [0062]    After incubation, the frame  102  is placed back on the base  100 , the cover removed, and flushed using vacuum. Alternatively, primary antibody can be recovered by extracting it from sample chamber  33  via port  38 , such as with a pipette. Any desired wash steps carried out by introducing wash solution to the reservoir  31 , allowing the sample  11  to soak (if needed) in the wash solution for the desired time, and then flushing by applying vacuum. A plurality of such wash steps can be employed if desired. 
         [0063]    Linker or secondary antibody of the desired concentration can be introduced into the injection/recovery port  38  in an amount sufficient to cover the sample  11 . If an incubation step is desired, the frame  102  is covered with cover  101 , and the task tracker set to the appropriate indication, such as “linker”. The covered frame  102  may then be incubated, such as at room temperature, in an incubator, or in a refrigerator for the desired time. 
         [0064]    After incubation, the frame  102  is placed back on the base  100 . Any desired wash steps may be carried out by removing the cover, introducing wash solution to the reservoir  31 , allowing the sample  11  to soak (if needed) in the wash solution for the desired time, and then flushing by applying vacuum. A plurality of such wash steps can be employed if desired. 
         [0065]    Reporter or tertiary antibody of the desired concentration can be introduced into the injection/recovery port  38  in an amount sufficient to cover the sample  11 . If an incubation step is desired, the frame  102  is covered with cover  101 , and the task tracker set to the appropriate indication, such as “reporter”. The covered frame  102  may then be incubated, such as at room temperature, in an incubator, or in a refrigerator for the desired time. 
         [0066]    After incubation, the frame  102  is placed back on the base  100 . Any desired wash steps may be carried out by removing the cover, introducing wash solution to the reservoir  31 , allowing the sample  11  to soak (if needed) in the wash solution for the desired time, and then flushing by applying vacuum. A plurality of such wash steps can be employed if desired. 
         [0067]    Chromogen can be introduced into the injection/recovery port  38  in an amount sufficient to cover the sample  11 . If an incubation step is desired, the frame  102  is covered with cover  101 , and the task tracker set to the appropriate indication, such as “chromogen”. The covered frame  102  may then be incubated, such as at room temperature, in an incubator, or in a refrigerator for the desired time. 
         [0068]    After incubation, the frame  102  is placed back on the base  100 . Any desired wash steps can be carried out by removing the cover, introducing wash solution to the reservoir  31 , allowing the sample  11  to soak (if needed) in the wash solution for the desired time, and then flushing by applying vacuum. A plurality of such wash steps can be employed if desired. 
         [0069]    Upon completion of the chromogen step, a final wash can be carried out before the slide holders are removed from the slide holder frame  102 . Removal of the slide  10  from the slide holder  30  can be carried out by bending away the bottom two compression clips  43  such as by inserting them into slots  66  in the slide holder assembly fixture  20  ( FIG. 13 ), thereby releasing the slide  10 . The slide holder  30  can be discarded. 
         [0070]      FIG. 14  illustrates another embodiment of a slide  10 ′. In the embodiment shown, the slide  10 ′ can be a molded or a diecut slide with a porous membrane  201  attached to it. The membrane can be attached to the slide by any suitable means, such as by heat sealing, ultrasonic welding, or with a suitable adhesive, such as silicone-based adhesive or an acrylic adhesive. A wide variety of membranes made from a wide variety of materials may be used in the embodiments described herein. Examples of such materials include polysaccharides, synthetic and semi-synthetic polymers, metals, metal oxides, ceramics, glass, and combinations thereof. Exemplary polymers that can be used to manufacture the membranes that may be used include, but are not limited to, substituted or unsubstituted polyacrylamides, polystyrenes, polymethacrylamides, polyimides, polyacrylates, polycarbonates, polymethacrylates, polyvinyl hydrophilic polymers, polystyrenes, polysulfones, polyethersulfones, copolymers or styrene and divinylbenzene, aromatic polysulfones, polytetrafluoroethylenes (PTFE), perfluorinated thermoplastic polymers, polyolefins, aromatic polyamides, aliphatic polyamides, ultrahigh molecular weight polyethylenes, polyvinylidene difluoride (PVDF), polyetheretherketones (PEEK), polyesters, and combinations thereof. Exemplary commercially available membranes are Durapore® and Millipore Express® and Millipore Express PLUS® available from EMD Millipore Corp. (Billerica, Mass.). Preferably the membranes are microporous membranes. 
         [0071]    Sample sections  301  such as tissue sections are mounted to the membrane  201 . The membrane  201  acts as a sample support, allowing blocking and antibody fluids, for example, to interact with the sample, but also allows for fluid transfer through the membrane  201  during rinsing and washing steps, for example. 
         [0072]    As shown in  FIGS. 15A ,  15 B and  15 C, each slide  10 ′ can be positioned directly into a corresponding slide well  302  in a vacuum manifold slide frame  205 . The vacuum manifold slide frame  205  may have one or more slide wells  302 . In certain embodiments, each slide well  302  has a gasket  401  such as a silicone or foam sealing gasket  401  to seal each slide  10 ′ in a respective slide well  302 . Upon application of vacuum as the driving force, fluid transfers through the membrane  301  and through a porous surface underdrain  501  beneath (in the direction of fluid flow) the membrane  201 . In certain embodiments, the underdrain includes molded drain slots, holes, sintered porous material, etc. that support the membrane due to membrane sagging. Preferably the underdrain is easy to clean but moldable and provides a vacuum driven draining from below. 
         [0073]      FIGS. 16A-16C  illustrate an alternative embodiment of a slide holder  30 ′. Like slide holder  30 , slide holder  30 ′ is a single use device and includes a reservoir  31 ′ and drain ports  41 ′,  41 ″ that respectively drain fluid from each chamber into drains  42 ′,  42 ″. Unlike slide holder  30 , slide holder  30 ′ includes multiple sample chambers  33 ′ and  33 ″ (two shown), each in fluid communication with the reservoir  31 ′ via narrow slit  32 ′ at the bottom of the reservoir  31 ′. In certain embodiments, the chambers  33 ′ and  33 ″ are opposed to one another and coextensive, but are in fluid isolation from one another so that each sample in a respective chamber is acted on independently of the other. In certain embodiments, each sample chamber  33 ′,  33 ″ includes a respective gasket  35 ′,  35 ″, such as a dispensed silicon gasket, as seen in  FIGS. 16B and 16C . In certain embodiments, the gaskets  35 ′,  35 ″ also could be overmolded, such as overmolded thermoplastic elastomer (TPE), overmolded silicone, diecut silicone, diecut double stick foam, thick diecut acrylic adhesive, etc. Each gasket  35 ′,  35 ″ includes a region that delimits a respective injection/recovery port fluid channel  36 ′,  36 ″. Each injection/recovery port fluid channel  36 ′,  36 ″ is in fluid communication with a respective injection/recovery port channel port  37 ′,  37 ″, which in turn is in fluid communication with a respective injection/recovery port  38 ′,  38 ″ configured to receive an injecting device such as a pipette. Each injection/recovery port fluid channel  36 ′,  36 ″ opens into a respective sample chamber  33 ′,  33 ″ at respective outlet openings  40 ′,  40 ″. 
         [0074]    In certain embodiments, the slides  10 ′,  10 ″ are attached to the slide holder  30 ′ in a manner similar to the above embodiment where only one slide and sample chamber is present. Suitable fluids can be introduced into the sample chamber  33 ′, such as via pipette or by the reservoir  31 ′. For example, the tips of respective pipettes can be inserted into the injection/recovery ports  38 ′,  38 ″. Fluid from each pipette flows into the respective injection/recovery port fluid channel  36 ′,  36 ″, respective outlet openings  40 ′,  40 ″ and into respective sample chambers  33 ′,  33 ″, where it contacts the samples. 
         [0075]      FIGS. 17A-17C  illustrate a horizontal embodiment where a tissue mounted substrate  10  such as a standard glass slide is constrained within a thermoformed slide holder flow cell  600  and sealed with a suitable adhesive  601 , such as a semi-permanent fluid-proof adhesive, such as an acrylic or silicone-based adhesive. In certain embodiments, the thermoformed slide holder flow cell  600  has a plurality of standoffs  602  positioned in each of the four corners to allow a slight gap on the sides and underneath the slide  10  to allow for drainage. The flow cell  600  includes an open top member  622  that is hingedly connected to slide holder member  623 . In certain embodiments, the hinge is a “living hinge” formed during the thermoforming process. The top member  622  is in the open position shown in  FIG. 17A  to load the slide into the flow cell  600 , and is then pivoted into a closed position as shown in  FIG. 17C . Various reagents, such as blocking agents, antibodies and stains can be placed onto the tissue on the slide  10 , and rinse and soak solutions can be introduced from the reservoir opening on the top  622  ( FIG. 17C ). 
         [0076]      FIGS. 18A-18C  illustrate an embodiment of a manifold suitable for use with the thermoformed slide holder flow cell  600  shown in  FIG. 17A . In certain embodiments, vacuum manifold frame  620  includes a plurality of slide holder wells  632 . Each well  632  is configured to receive a respective slide holder flow cell  600 . In certain embodiments, each well  632  includes a drain port  624 , which receives the drain  603  of the slide holder flow cell  600  in sealing relation ( 627 ). In certain embodiments, a drain channel  626  is formed in the manifold frame  620  in communication with the drain port  624 . Upon the introduction of a driving force such as vacuum, fluid is flushed from the slide through the multiple bypasses  605 , and down through the drain  603  and then through the drain channel  626 . A valve, such as a duckbill valve, may be positioned in the port  624  and prevents fluid from exiting until the valve is overcome by the application of vacuum. 
         [0077]      FIG. 19  illustrates an embodiment where the adhesive  640  on the underside of the thermoformed slide holder flow cell  600  seals directly to the slide  10  surface. A small slit  645  in the adhesive  640  acts as a drain port. Soaking and rinsing fluids are introduced using the reservoir opening  630 , allowing the flushing of compounds off the tissue section via vacuum on a vacuum manifold. 
         [0078]      FIGS. 20A-20C  illustrate a thermoform vertical embodiment. A standard tissue mounted glass slide  10  is constrained within a thermoformed slide holder flow cell  200 , sealed with a semi-permanent fluid-proof adhesive  260 , such as a silicone-based adhesive or acrylic adhesive. In certain embodiments, the thermoform has standoffs  400  in all four corners to allow a slight gap on the tissue side of the slide  10 . A plurality of spaced longitudinal ribs  411  can be provided to provide added rigidity to the flow cell  200 , and to provide a gap allowing proper drainage upon application of a driving force. When assembled, the thermoform  200  creates a gap in front of the tissue that allows reagents such as blocking agents, antibodies, stains, etc. to be in contact with the tissue, although rinse and soak solutions, for example, can be introduced from the reservoir  203  opening on the top. When a driving force such as vacuum is applied, the fluid is flushed from the slide through the capillary drain (e.g., a small slit)  500  in the adhesive, and down through a drain. In certain embodiments, the thermoform is formed from a first member  401  pivotally connected to a second member  402  by a hinge  403  to allow for quick assembly. Although in the embodiment shown the hinge  403  is shown along the longitudinal axis of the flow cell, in certain embodiments the hinge could be oriented at the base of the flow cell. 
         [0079]    As shown in  FIGS. 21 and 22 , the tip of a pipette  39  may introduce fluids such as antibodies and wash fluids, into the reservoir  203 , the reservoir  203  being in fluid communication with the tissue chamber  204  that includes a known region for the fluids. The flow cell  200  is shown positioned in a vacuum manifold sealing assembly. In certain embodiments, the vacuum manifold  100 ′ includes one or more flow cell receiving wells  149 ′ that can include silicone tubing  320 ,  321  or the like. In certain embodiments, the flat thermoform slips between two tubes  320 ,  321 , creating a seal around the thermoform assembly. When a driving force such as vacuum is applied, fluid drains out of the capillary drain  500  in the adhesive layer created by the sandwich of the thermoform and the adhesive, as illustrated by the dotted arrow  510  in  FIGS. 21 and 22 .