IN SITU HEAT INDUCED ANTIGEN RECOVERY AND STAINING METHOD

Abstract

An automated in situ heat induced antigen recovery and staining method and apparatus for treating a plurality of microscope slides. The process of heat induced antigen recovery and the process of staining the biological sample on the microscope slide are conducted in the same apparatus, wherein the microscope slides do not need to be physically removed from one apparatus to another. The reaction conditions for treating a slide can preferably be controlled independently, including the individualized application of reagents to each slide and the individualized treatment of each slide.

Claims

1. A method of treating biological samples, comprising: placing a first microscope slide on a first slide support element with the first microscope slide having a first biological sample disposed thereon; moving the first slide support into a treatment chamber of an antigen recovery and staining apparatus so as to position the first microscope slide and the first biological sample in the treatment chamber; treating the first biological sample by applying a first reagent to the first biological sample, heating the first reagent to a desired temperature for a desired period of time, and applying additional reagents of a treatment protocol; placing a second microscope slide position on a second slide support element with the second microscope slide having a second biological sample disposed thereon; moving the second slide support into the treatment chamber of the antigen recovery and staining apparatus so as to position the second microscope slide and the second biological sample in the treatment chamber while the first biological sample continues to be treated in the treatment chamber; treating the second biological sample by applying a second reagent to the second biological sample, heating the second reagent to a desired temperature for a desired period of time, and applying additional reagents of another treatment protocol; determining if one of the first biological sample and the second biological sample has completed the treatment protocol; and removing the microscope slide with the biological sample that has completed the treatment protocol from the treatment chamber while the other biological sample continues to be treated in the treatment chamber, wherein the step of removing the microscope slide with the biological sample that has completed the treatment protocol from the treatment chamber comprises the steps of: moving the slide support out of the treatment chamber of the antigen recovery and staining apparatus so as to position the microscope slide with the biological sample that has completed the treatment protocol outside of the treatment chamber; and removing the microscope slide with the biological sample that has completed the treatment protocol from the slide support element.

2. The method of claim 1 further comprising the steps of: positioning the first biological sample in a first reaction compartment and positioning the second biological sample in a second reaction compartment.

3. The method of claim 2, wherein the first reaction compartment is separate from the second reaction compartment.

4. The method of claim 2, wherein the step of positioning the first biological sample in the first reaction compartment, the first microscope slide defines a portion of the first reaction compartment, and wherein the step of positioning the second biological sample in the second reaction compartment, the second microscope slide defines a portion of the second reaction compartment.

5. The method of claim 2, wherein the step of positioning the first biological sample in the first reaction compartment, the first slide support element defines a portion of the first reaction compartment, and wherein the step of positioning the second biological sample in the second reaction compartment, the second slide support element defines a portion of the second reaction compartment.

6. The method of claim 1, further comprising the steps of: placing a third microscope slide on the slide support element from which the microscope slide with the biological sample that has completed the treatment protocol has been removed with the third microscope slide having a third biological sample disposed thereon; and moving the slide support with the third microscope slide positioned thereon into the antigen recovery and staining apparatus so as to position the third microscope slide and the third biological sample in the antigen recovery and staining apparatus while the other biological sample continues to be treated in the antigen recovery and staining apparatus.

7. The method of claim 1, further comprising: applying at least one of the first reagent and the additional reagents of the treatment protocol from a first reagent dispensing pack, the first reagent dispensing pack having at least one individually contained reagent to be applied to the first biological sample; and applying at least one of the second reagent and the additional reagents of the other treatment protocol from a second reagent dispensing pack, the second reagent dispensing pack having at least one individually contained reagent to be applied to the second biological sample.

8. The method of claim 7, wherein each of the at least one individually contained reagent in the first reagent dispensing pack and the second reagent dispensing pack comprises a single dose for application to a single microscope slide.

9. A method of treating biological samples, comprising: placing a first microscope slide on a first slide support element with the first microscope slide having a first biological sample disposed thereon; placing a second microscope slide on a second slide support element with the second microscope slide having a second biological sample disposed thereon; moving the first slide support into an antigen recovery and staining apparatus; moving the second slide support into the antigen recovery and staining apparatus; treating the first biological sample by applying a first reagent to the first biological sample, heating the first reagent to a desired temperature for a desired period of time, and applying additional reagents of a treatment protocol; treating the second biological sample by applying a second reagent to the second biological sample, heating the second reagent to a desired temperature for a desired period of time, and applying additional reagents of another treatment protocol; determining if one of the first biological sample and the second biological sample has completed the treatment protocol; and removing the microscope slide with the biological sample that has completed the treatment protocol from the antigen recovery and staining apparatus while the other biological sample continues to be treated in the antigen recovery and staining apparatus, wherein the step of removing the microscope slide with the biological sample that has completed the treatment protocol from the antigen recovery and staining apparatus comprises the steps of: moving the slide support out of the antigen recovery and staining apparatus so as to position the microscope slide with the biological sample that has completed the treatment protocol outside of the antigen recovery and staining apparatus; and removing the microscope slide with the biological sample that has completed the treatment protocol from the slide support element.

10. The method of claim 9 further comprising the steps of: positioning the first biological sample in a first reaction compartment and positioning the second biological sample in a second reaction compartment.

11. The method of claim 10, wherein the first reaction compartment is separate from the second reaction compartment.

12. The method of claim 10, wherein the step of positioning the first biological sample in the first reaction compartment, the first microscope slide defines a portion of the first reaction compartment, and wherein the step of positioning the second biological sample in the second reaction compartment, the second microscope slide defines a portion of the second reaction compartment.

13. The method of claim 10, wherein the step of positioning the first biological sample in the first reaction compartment, the first slide support element defines a portion of the first reaction compartment, and wherein the step of positioning the second biological sample in the second reaction compartment, the second slide support element defines a portion of the second reaction compartment.

14. The method of claim 9, further comprising the steps of: placing a third microscope slide on the slide support element from which the microscope slide with the biological sample that has completed the treatment protocol has been removed with the third microscope slide having a third biological sample disposed thereon; and moving the slide support with the third microscope slide positioned thereon into the antigen recovery and staining apparatus so as to position the third microscope slide and the third biological sample in the antigen recovery and staining apparatus while the other biological sample continues to be treated in the antigen recovery and staining apparatus.

15. The method of claim 9, further comprising: applying at least one of the first reagent and the additional reagents of the treatment protocol from a first reagent dispensing pack, the first reagent dispensing pack having at least one individually contained reagent to be applied to the first biological sample; and applying at least one of the second reagent and the additional reagents of the other treatment protocol from a second reagent dispensing pack, the second reagent dispensing pack having at least one individually contained reagent to be applied to the second biological sample.

16. The method of claim 15, wherein each of the at least one individually contained reagent in the first reagent dispensing pack and the second reagent dispensing pack comprises a single dose for application to a single microscope slide.

17. A method of treating biological samples, comprising: placing a first microscope slide on a first slide support element with the first microscope slide having a first biological sample disposed thereon; moving the first slide support into an antigen recovery and staining apparatus; treating the first biological sample by applying a first reagent to the first biological sample, heating the first reagent to a desired temperature for a desired period of time, and applying additional reagents of a treatment protocol; placing a second microscope slide on a second slide support element with the second microscope slide having a second biological sample disposed thereon; moving the second slide support into the antigen recovery and staining apparatus so as to position the second microscope slide and the second biological sample in the antigen recovery and staining apparatus while the first biological sample continues to be treated in the antigen recovery and staining apparatus; treating the second biological sample by applying a second reagent to the second biological sample, heating the second reagent to a desired temperature for a desired period of time, and applying additional reagents of another treatment protocol; determining if one of the first biological sample and the second biological sample has completed the treatment protocol; and removing the microscope slide with the biological sample that has completed the treatment protocol from the treatment chamber while the other biological sample continues to be treated in the treatment chamber, wherein the step of removing the microscope slide with the biological sample that has completed the treatment protocol from the antigen recovery and staining apparatus comprises the steps of: moving the slide support out of the antigen recovery and staining apparatus so as to position the microscope slide with the biological sample that has completed the treatment protocol outside of the antigen recovery and staining apparatus; and removing the microscope slide with the biological sample that has completed the treatment protocol from the slide support element.

18. The method of claim 17 further comprising the steps of: positioning the first biological sample in a first reaction compartment and positioning the second biological sample in a second reaction compartment.

19. The method of claim 18, wherein the first reaction compartment is separate from the second reaction compartment.

20. The method of claim 18, wherein the step of positioning the first biological sample in the first reaction compartment, the first microscope slide defines a portion of the first reaction compartment, and wherein the step of positioning the second biological sample in the second reaction compartment, the second microscope slide defines a portion of the second reaction compartment.

21. The method of claim 18, wherein the step of positioning the first biological sample in the first reaction compartment, the first slide support element defines a portion of the first reaction compartment, and wherein the step of positioning the second biological sample in the second reaction compartment, the second slide support element defines a portion of the second reaction compartment.

22. The method of claim 17, further comprising the steps of: placing a third microscope slide on the slide support element from which the microscope slide with the biological sample that has completed the treatment protocol has been removed with the third microscope slide having a third biological sample disposed thereon; and moving the slide support with the third microscope slide positioned thereon into the antigen recovery and staining apparatus so as to position the third microscope slide and the third biological sample in the antigen recovery and staining apparatus while the other biological sample continues to be treated in the antigen recovery and staining apparatus.

23. The method of claim 17, further comprising: applying at least one of the first reagent and the additional reagents of the treatment protocol from a first reagent dispensing pack, the first reagent dispensing pack having at least one individually contained reagent to be applied to the first biological sample; and applying at least one of the second reagent and the additional reagents of the other treatment protocol from a second reagent dispensing pack, the second reagent dispensing pack having at least one individually contained reagent to be applied to the second biological sample.

24. The method of claim 23, wherein each of the at least one individually contained reagent in the first reagent dispensing pack and the second reagent dispensing pack comprises a single dose for application to a single microscope slide.

Description

DESCRIPTION OF THE DRAWINGS

[0011] FIG. 1 is a perspective view of an apparatus of the invention (shown without a pressing element for crushing a reagent capsule).

[0012] FIG. 2 is a cross-sectional view of the apparatus of FIG. 1 (shown with a pressing element for crushing a reagent capsule).

[0013] FIG. 3A is a cross-sectional view of the apparatus of FIG. 1 (shown with a reaction compartment having a raised slide support surface) taken through line 3A-3A.

[0014] FIG. 3B is a cross-sectional view of the apparatus of FIG. 1 (shown with a reaction compartment having a lowered slide support surface) taken through line 3B-3B.

[0015] FIG. 4 is a cross-sectional view of an alternative embodiment of the apparatus of the present invention having an alternate type of slide support surface.

[0016] FIG. 5 is a perspective view of a reagent strip of the present invention.

[0017] FIG. 6 is a cross-sectional view of the reagent strip of FIG. 5 taken through the line 6-6.

[0018] FIG. 7 is an elevational view of a modular apparatus containing a plurality of the apparatus of FIG. 1.

[0019] FIG. 8 is a flow chart showing a preferred sequence of steps in the method of the present invention.

[0020] FIG. 9 is a schematic view of an apparatus of the invention and a microprocessor which controls the apparatus.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention is directed to an automated method and apparatus for treating biological samples on microscope slides for unmasking (retrieving or recovering) epitopes or antigens of the biological samples and then staining or otherwise treating the biological samples. The automated apparatus comprises an array of individual reaction compartments, each of which is used to treat a single microscope slide (also referred to herein as a slide), wherein each reaction compartment preferably can function and can be controlled independently of the other reaction compartments in the array. Each reaction compartment in the array comprises a support element comprising a surface upon which a microscope slide can be supported and positioned adjacent or inserted into the compartment for treatment with a reagent. The support element further comprises, in a preferred embodiment, a conduction type heating element for heating the microscope slide to a predetermined treatment temperature when desired. The support element with the microscope slide thereon can be raised into or adjacent the reaction compartment for treatment of the microscope slide, or lowered or removed from the reaction compartment for placement of a microscope slide onto or removed from the support surface or for removal of a reagent or rinsing solution from the microscope slide during the treatment process.

[0022] Reagents, such as antibodies, enzymes, rinse buffers, antigen recovery buffers, or stains, are contained in an individualized reagent dispensing strip which is specific for each microscope slide to be treated. Since each microscope slide and reaction compartment is generally provided with its own reagent dispensing strip, each microscope slide can be treated independently with a different set of reagents (a particular treatment protocol) while being treated simultaneously with other microscope slides. Similarly, in an especially preferred embodiment of the invention, each microscope slide can be heated separately, as well as treated with a different treatment protocol. The apparatus of the present invention therefore comprises, in a preferred embodiment, a plurality of individualized reaction compartments in a chamber which can be substantially closed for minimizing evaporation during heating. A microscope slide can be supported in each reaction compartment, and each microscope slide can be heated separately therein. A reagent dispensing strip containing a plurality of individually contained reagents (reagent bubbles, blisters or capsules) is positioned upon an upper portion of each reaction compartment, and at an appropriate time, a reagent from each reagent dispensing strip is expelled from a reagent capsule under compression and is thereby applied to the biological sample on the microscope slide. Or, a reagent, such as an antigen recovery buffer can be introduced via a separate dispenser. The term reagent is defined herein to include any type of fluid material that may be applied to the biological material on the microscope slide, including antibodies, stains, enzymes, buffers, rinses, or washes, or any other material applied in the process of antigen recovery or treating the biological material on the microscope slide to be viewed under the microscope.

[0023] During an antigen recovery step, the microscope slide, sample, and antigen recovery buffer thereon are heated to an appropriate temperature for a predetermined duration to cause the antigen recovery buffer to react with the sample on the microscope slide, after which the antigen recovery buffer is removed from the microscope slide, preferably by washing or flooding the microscope slide or chamber containing the microscope slide with a rinse buffer and allowing the rinse buffer to drain off by gravity or by blowing the solution off the microscope slide using pressurized air. Each microscope slide may be treated in the same manner, or may be treated with different reagents using a different treatment protocol, preferably simultaneously, yet independently.

[0024] When a reagent is provided via a reagent dispensing strip, the apparatus is preferably equipped with a drive mechanism for causing the reagent dispensing strip to be advanced in a forward direction wherein each reagent capsule in succession is positioned above an aperture in the compartment through which the reagent in the capsule is delivered. The reagent dispensing strip may be advanced using rollers positioned along the upper end of the compartment or a pushing mechanism which pushes upon the rear end of the reagent dispensing strip. The reagent in the reagent capsule of the reagent dispensing strip is to be applied to the microscope slide by a pressing mechanism which, in a preferred version, compresses and thereby crushes the reagent capsule and causes the reagent to be expelled and deposited directly onto the microscope slide.

[0025] In a preferred method of the present invention, a plurality of microscope slides, each having thereon a sample to be treated, is provided. Each microscope slide is positioned upon a support element which is then moved into an application position. A plurality of reagent dispensing strips is provided, one for each microscope slide to be treated. Each microscope slide is subjected to an antigen recovery step then is treated by applying a reagent from its corresponding reagent dispensing strip. Each microscope slide can be handled differently, if desired, during the treatment cycle. After a predetermined duration, the microscope slide and support element is moved to a removal position wherein the reagent is removed, preferably in between reagent applications, by treatment with a rinsing solution to remove the reagent prior to further treatment. Each microscope slide can be treated according to the treatment protocol specific to that sample or that particular microscope slide. All microscope slides may be treated using the same protocol, or one or more, or all, of the microscope slides may be treated using a different protocol.

[0026] An example of a treatment protocol comprises:

[0027] 1) antigen recovery, 10 minutes at 98 C.,

[0028] 2) cool, 20 minutes,

[0029] 3) rinse buffer,

[0030] 4) primary antibody, 30 minutes,

[0031] 5) rinse,

[0032] 6) biotinylated linking antibody, 10 minutes,

[0033] 7) rinse buffer,

[0034] 8) peroxidase labeled streptavidin label,

[0035] 9) rinse buffer,

[0036] 10) 3,3-diaminobenzidine chromogen,

[0037] 11) rinse buffer,

[0038] 12) chromogen enhancer,

[0039] 13) rinse buffer, and

[0040] 14) counter stain.

[0041] A variety of other treatment protocols are well known to those of ordinary skill in the art and further discussion of them herein is not deemed necessary. Each microscope slide, if necessary, may be heated prior to application of the reagent, if necessary, then may be cooled as the reagent is removed, then reheated, if necessary, prior to or after addition of the next reagent. The entire process is run automatically once the microscope slide is disposed onto the support element, and the reagent dispensing strip is positioned upon the upper side of the reaction compartment.

[0042] Turning now to the drawings, a specific embodiment of the apparatus of the present invention is shown in FIGS. 1-6. Although FIGS. 1-6 show a preferred version of the invention, it will be understood that the embodiment shown in FIGS. 1-6 is but one of many possible versions of the apparatus enabled herein which will come to the mind of a person of ordinary skill in the art.

[0043] Shown in FIG. 1, and designated therein by the general reference numeral 10 is an antigen recovery and staining apparatus constructed in accordance with the present invention. The antigen recovery and staining apparatus 10 comprises a treatment chamber 12 which further comprises a plurality of reaction compartments 14 (see FIGS. 2-4). Preferably the treatment chamber 12 generally comprises from 10 to 20 reaction compartments 14 but may contain more or fewer. Each reaction compartment 14, when enclosed, minimizes evaporation of a reagent solution when a microscope slide is exposed to high temperature pretreatment conditions. Each reaction compartment 14 has an upper side 16 having an opening 18 therein, a lower side 20, and a pair of sidewalls 22 which extend from the rear end 23a of the treatment chamber 12 to the front end 23b of the treatment chamber 12. Positioned above each reaction compartment 14 is a reagent dispensing strip holder 24 for holding and guiding a reagent dispensing strip 26 (see FIGS. 5 and 6). Each reagent dispensing strip 26 has a front end 28 and a rear end 30 and a plurality of capsules 32 made of a crushable plastic material such as polyethylene or another suitable material (e.g., polypropylene or polystyrene) and which may include one or more multiple capsules 32a. The size of each capsule 32 or multiple capsule 32a may be adjusted to accommodate the amount of reagent which is desired to be applied to a microscope slide 44. Each capsule 32 or multiple capsule 32a contains a reagent or treatment solution which is intended to be applied to a biological material on the microscope slide 44. Multiple capsule 32a is useful in a method wherein two or more reagents must be contained separately before being applied to the microscope slide 44. When the multiple capsule 32a is crushed by the pressing mechanism 36, two or more reagents contained within the capsule 32a are combined and simultaneously applied to the microscope slide 44.

[0044] Other embodiments of the reagent dispensing strip 26 and the reagent capsule 32 and multiple capsule 32a will readily be apparent to one of ordinarily skill in the art. For example, each reagent dispensing strip 26 may comprise a one or more blank spaces for insertion of individualized capsules 32 by a user. Below each capsule 32 or multiple capsule 32a is an aperture or weak area 34 in the reagent dispensing strip 26 through which the reagent in the capsule 32 or multiple capsules 32 can be forced by a pressing mechanism 36. The blank space or space left by the puncturing of a capsule 32 or 32a, or vents in the reagent dispensing strip 26 may function to release pressure, steam or vapors produced during the treatment process. The reagent dispensing strip 26 is advanced in a direction 37 toward the front end 23b of the treatment chamber 12 by a reagent strip drive mechanism 38 driven, for example, by an electric motor which in FIGS. 1, 3A and 3B is shown as a pushing mechanism comprising a threaded shaft, but which may instead by a mechanism (not shown) comprising rollers which drive, draw or pull the reagent strip holder 24 in a forward direction 37.

[0045] Each reaction compartment 14 further comprises at its lower side 20 a slide support element 40 having a slide tray 42 upon which the microscope slide 44 can be positioned and held for treatment. The slide support elements 40 together comprise a slide support assembly 39. With the microscope slide 44 disposed on the slide support element 40, the slide support element 40 and the microscope slide 44 are positioned in an application position to fit adjacent the lower side 20 of the reaction compartment 14, thereby constituting an openable bottom of the reaction compartment 14. The slide support element 40 further has a heating element 46 incorporated therein for heating the microscope slide 44 as discussed elsewhere herein. In one embodiment, the slide support element 40 has a hinge 48 for enabling the slide support element 40 to be moved (raised) into an application position (FIG. 3A) and therefrom lowered (e.g., tilted) into an opened position (see FIG. 3B). Alternatively, the slide support element 40 may be raised and lowered into position by another mechanism, such as a stepper motor 58 and screw drive 59 mechanism (FIG. 4). Each reaction compartment 14 further comprises a manifold 50 which comprises, in a preferred embodiment, a plurality of reagent dispensing ports or elements including, for example but not limited to, an antigen recovery buffer dispenser 51 connected via an antigen recovery buffer supply line 51a to an antigen recovery buffer supply (not shown), a rinse buffer dispenser 52 connected via a rinse buffer supply line 52a to a rinse buffer supply (not shown) and an air pressure nozzle (pressurized air nozzle) 54 connected via an air line 54a to an air supply (not shown). The antigen recovery buffer dispenser 51 applies an antigen recovery buffer to the microscope slide 44 for the antigen recovery treatment step prior to staining or other preparation of the biological material on the microscope slide 44. The rinse buffer dispenser 52 applies a rinse buffer 56 to the microscope slide 44 to rinse a reagent from the microscope slide 44. The air pressure nozzle (pressurized air nozzle) 54 functions to clear away a rinse buffer 56 from the microscope slide 44. Dispensers 51 and 52 may be used to dispense other reagents, and may constitute more than, or fewer than, the dispensers shown in FIGS. 2, 3A, 3B, and 4. The microscope slide 44 is generally disposed in a removal position for facilitating removal of the rinse buffer 56 as shown in FIGS. 1 and 3B. Each slide support element 40, in a preferred embodiment, can be heated or moved independently of any other slide support element 40, although one of ordinary skill in the art can envision that the slide support elements 40 may be designed to operate in concert, i.e., simultaneously. Each reaction compartment 14 preferably can contain a volume of up to 15 ml. The slide support element 40 and the microscope slide 44 supported thereon may be in a horizontal position (e.g., FIGS. 1, 2, 3A and 4) or in a tilted position (e.g., FIGS. 1 and 3B).

[0046] The antigen recovery and staining apparatus 10 can be controlled automatically wherein predetermined sequences and operations are carried out using various electromechanical systems which are not shown but which are well known to those of ordinary skill in the art. For example, each of the steps of raising into a treatment position and lowering into a removal position each of the slide support elements 40, applying an antigen recovery buffer, advancing each reagent dispensing strip 26, compressing each capsule 32 or 32a of the reagent dispensing strip 26, heating each microscope slide 44 on the slide support surface 40, applying a rinse buffer 56 to the microscope slide 44, removing the rinse buffer 56 or other reagent from the microscope slide 44, and treating each microscope slide 44 independently can be automatically controlled and programmed using programming methods and devices well known in the art. Because each reaction compartment 14 and slide support element 40 can be controlled independently, a microscope slide 44 can even be removed or inserted even while other reaction compartments 14 are in operation.

[0047] Preferably, a microprocessor 62, controls the antigen recovery and staining apparatus 10 as shown in FIG. 9. That is, an operator programs the microprocessor 62 with information such as which reaction compartments 14 are to be used and to what temperature each is to be heated and at which steps, then programs the particular treatment protocol to be performed on the sample on each microscope slide 44 on each slide support element 40. Variables in these protocols can include the particular type of reagent dispensing strip 26 to be used, the time that each reagent or treatment solution on the reagent dispensing strip 26 will be allowed to react with the sample on the microscope slide 44, whether the microscope slide 44 will be heated, and if so to what temperature and for how long, and the manner in which the microscope slide 44 will be rinsed, for example. Other variables not listed herein may also be programmed.

[0048] The invention may further comprise a modular apparatus 60 comprising a plurality of antigen recovery and staining apparatuses 10 each serving as an individual module in the modular apparatus 60. The individual modules can be stacked together for example, as shown in FIG. 7, or may be oriented in any other desirable manner.

[0049] Shown in FIG. 8 is a schematic drawing which describes the preferred method of the present invention. In the first step, a microscope slide 44 which has a sample disposed thereon is provided, and is disposed onto a slide support element 40 which is moved into an application or treatment position adjacent or against the reaction compartment 14. If a plurality of microscope slides 44 are supplied, each microscope slide 44 is disposed on a separate microscope slide support element 40 and the microscope slides 44 are moved independently or simultaneously into an application position.

[0050] Once in the application position, an antigen recovery buffer is initially applied to the sample on the microscope slide 44. Microscope slide 44 is then heated to a desired, predetermined temperature, for example from about 120 C. to about 160 C. whereby the antigen recovery buffer is heated to a temperature of from about 90 C. to 100 C., for example. The microscope slide 44 is allowed to react with the reagent for a predetermined length of time, for example, 10 to 30 minutes, preferably at 9598 C. Venting of steam may occur through small holes (not shown) in the reagent strip 26 or elsewhere in the reaction compartment 14. It is not necessary to add additional antigen recovery buffer during this step. After the reaction period is over, the slide support element 40 and the microscope slide 44 thereon are moved (lowered or dropped) to a removal position, if necessary, where the antigen recovery buffer is removed from the microscope slide 44, for example, by applying a rinsing solution or buffer to the microscope slide 44 or by gravity or by pressurized air. A rinse solution or buffer may be applied and removed more than once for treatment or for removal of a particular reagent before or after lowering the microscope slide 44 to the removal position. It may be desirable to add rinse buffer to the microscope slide 44 to cool the microscope slide 44 prior to lowering the microscope slide 44 to the removal position, for example, by adding rinse buffer 56 to the antigen recovery buffer before the microscope slide 44 is moved to the removal position. After the microscope slide 44 has been treated for antigen recovery, another reagent can then be applied for treatment of the sample on the microscope slide 44. In this step, the microscope slide 44 and slide support element 40 are then returned to the application position, a reagent is applied, and is then removed after the treatment period. The series of steps may be repeated. When the treatment of the sample is completed, the microscope slide 44 is removed from the slide support element 40 for further treatment or analysis apart from the antigen recovery and staining apparatus 10.

[0051] Changes may be made in the construction and the operation of the various components, elements and assemblies described herein or in the steps or the sequence of steps of the methods described herein without departing from the scope of the invention as defined in the following claims.