WO2003019148A1

WO2003019148A1 - Method and device for staining tissues

Info

Publication number: WO2003019148A1
Application number: PCT/GB2002/003744
Authority: WO
Inventors: Krishna Datla
Original assignee: Imperial College Innovations Limited
Priority date: 2001-08-24
Filing date: 2002-08-14
Publication date: 2003-03-06
Also published as: GB0120612D0

Abstract

A method of staining a tissue section comprising the steps of (i) providing a fixed tissue section; (ii) placing the tissue section of step (i) into a container with at least one opening and at least one perforation wherein the container is at least 0.75 cm in width or diameter or depth but less than 10 cm in width or diameter or depth wherein the at least one perforation has a maximum dimension of 2 mm; (iii) placing the container comprising the tissue section in a vessel containing a first solution such that the solution in the vessel enters the container via the perforations in the container; (iv) incubating the tissue section in the first solution; and (v) removing from the container at least some of the solution via the perforations in the container. The invention also provides a device useful in said method.

Description

METHOD AND DEVICE FOR STAINING TISSUES

The present invention relates to methods for staining tissue sections and devices for use in the methods.

An established method of analysing biological tissue is to slice a fine section from the tissue, stain the tissue section and then view the stained section, for example, under a microscope. Tissue stains generally alter the optical characteristics of a tissue to facilitate imaging. A range of tissue stains are known, and many of these differ from each other in terms of the component in the tissue that they bind to. Immunohistochemistry is a specific method of staining a component in a tissue section involving an antibody, which is selective for the component of interest, or a particular conformation of the component of interest, as part of the stain.

The staining procedure of immunohistochemistry usually involves incubating the tissue section sequentially with several reagents, such as the antibody itself and any washing solutions which remove excess antibody from the tissue section.

The thinner the tissue section used, then the more clearly the details of any staining can be observed. However, the tissue sample used to generate sections are usually small, and increasing the thinness of the tissue section means that extremely small and delicate pieces of tissue have to be manipulated. Transferring such small sections between solutions cannot be done using a pair of, for example, a pair of tweezers, since this risks damaging the section. Generally, transfer of tissue sections between solutions during staining is done by lifting the section using a paintbrush, to ensure that the sections are treated gently. Clearly, manipulating a tissue section with a paintbrush requires a steady hand and can take time, since each tissue section must be transferred individually between solutions.

The requirement for such individual treatment, care and inter-incubation handling time accounts for a considerable proportion of the overall time taken to produce a stained tissue section, and restricts the number of sections which can be handled at any one time.

The inventor has now developed a new method of manipulating tissue sections during staining. This method is easier than current tissue section staining methods, reduces the time taken between incubations and permits bulk handling of tissue sections. The invention provides and makes use of a sieve-like device which can retain the tissue sections and permit fluid entry into, and exit from, the device.

A first aspect of the invention provides a method of staining a tissue section comprising the steps of

(i) Providing a tissue section; (ii) Placing the tissue section of step (i) into a container with at least one opening and at least one perforation wherein the container is at least 0.75cm in width or diameter or depth but less than 10cm in width or diameter or depth wherein the at least one perforation has a maximum dimension of 2mm; (iii) Placing the container comprising the tissue section in a vessel containing a first solution such that at least some of the solution in the vessel enters the container via the at least one perforation in the container; (iv) Incubating the tissue section in the first solution; and (v) Removing from the container at least some of the solution via the perforations in the container.

It will be appreciated that the first solution does not have to be in the vessel when the container first enters it; the solution may be added to the vessel after the container is placed into it. Similarly, the vessel may contain some of the first solution, and more solution may be added once the container has been placed into the vessel. For ease of manipulation, it is preferred that at least some of the solution is already in the vessel when the container is placed into it. More preferably, all of the first solution is in the vessel before the container is placed into it.

By "tissue section" we include tissue slices of any convenient thickness or dimension.

The tissue section may be a section from any biological tissue source. Examples of suitable tissues include brain, kidney, liver, pancreas, bone, adipose, muscle, testis, ovary, placenta and the like. The tissue source may well require preparation to make it suitable for sectioning. Typical preparation methods include perfusion or fixing with chemicals such as paraformaldehyde followed by cryo-protecting with different concentrations of sucrose. The tissue can then be mounted on a tissue holder in the cryostat for sectioning.

Methods of cutting, or "sectioning" tissues are well known in the art of biology. . The tissue may be sectioned into slices which are any convenient thickness. Preferably, the sections are between 5 microns and 100 microns in thickness, more preferably between 10 and 30 microns, and still more preferably, the sections are between 15 and 20 microns in thickness. It will be appreciated that the method may be applicable to other manipulations of tissue sections, such as de-hydration, de-waxing, de- fatting and any other general cleaning procedures.

Preferably, the tissue section used in the method is fixed. Methods of fixing tissues are well known in the art, and include incubation with formalin, paraformaldehyde and glutaraldehyde. Also preferably, the tissue section is cryo-protected. Hence, the tissue section may be fixed and/or cryo- protected before use in the method.

The container which is useful in the method of the invention is any container which can act as a sieve, capable of retaining the tissue sections within the container but allowing the free passage of a liquid or solution through the perforations in the container.

Preferably, the container is made from a material which does not, or does only to a small extent, adsorb proteins or other biological molecules onto its surface when placed in contact with a solution of the biological molecule. By "small extent" we mean that less than 50 000, 10 000, 5 000, 1 000, 500 biological molecules are adsorbed per mm² of the material surface, preferably less than 500, 250, 100, 50 or 10 or 1 biological molecules are adsorbed per mm .

In practical terms, it may be easier to determine the degree of adsorption in terms of the drop in concentration of the solution which is in contact with the material. Hence, preferably the container is one which, when in contact with the solution used in the method, does not cause a drop in concentration (by adsorption of molecules from the solution onto the material) of the solution of greater than 50%, 40%, 30%, 20% or 10%. Preferably, any drop in concentration of the solution is less than 5%, 3% or 1%. More preferably, any drop in concentration is negligible, or undetectable.

It is preferred if the container is further made from a material which does not rust or chemically react with solutions generally used in immunohistochemical staining. Hence, it is preferred if the container is made from a chemically inert material. Preferably, the container is made from a plastic or glass. More preferably, the container is made from transparent plastic or glass, and still more preferably, the container is made from polymethyl methacrylate (Perspex™).

The walls of the container may be any convenient thickness. It is preferred if the thickness of the walls of the container is less than 1.5mm, more preferably less than 1mm. An advantage of the walls of the container being thinner than 1.5mm or 1mm is that fluids may enter and exit via the perforations more easily. This is especially useful where the fluid is a viscous fluid, such as a protein solution of, for example, more than 1% or 10%.

The container may be any convenient shape or form. Preferably, the sides of the container are straight in at least one plane. Preferably, the container is cylindrical, for example a cylinder with one open end and one closed end (which closed end may be perforated). Preferably, the base of the cylinder is such that it allows the container to stand stably on its base. More preferably, the base is substantially flat, with the sides of the cylinder being perpendicular to the base. Alternatively, the container may be substantially frustroconical in shape.

The advantage of a cylindrical shape is that it allows the container to be inserted into a vessel, such as a laboratory beaker, by lowering the container into an opening in the vessel. Preferably, insertion of the container into a vessel is such that the walls of the container and vessel become closely associated. The vessel may be cylindrical (or frustroconical) with an open end (through which the container may be inserted) and a closed end (so that the vessel is capable of retaining liquid). By "closely associated" we mean that when the container is inside the vessel, the maximum distance between a wall of the vessel and a wall of the container is less than 50mm, preferably less than 25mm or 10mm and still more preferably, less than 5mm, 4mm or 3mm. Suitable vessels are described in more detail below, and an example is shown in Figure 4.

Preferably, at least some of the solution in the container is removed in step (v) by removing, including partially removing, the container from the vessel, for example, by raising the container in the vessel. Alternatively, the solution may be removed by removing the solution from the vessel. In this case, providing the container has a perforation in a suitable position (eg, below the solution level in the vessel once the container is inside the vessel), then as solution is removed from the vessel, the level of solution in the vessel drops, and solution may exit from the container into the vessel.

It is not necessary that all the solution in the container exits the container by means of the ρerforation(s). However, it is preferred if all the solution in the container exits via the perforations. It is preferred if the solution does not exit by the opening of the container.

By "perforation" we mean a small hole or aperture in the walls of the container through which a liquid can pass from inside the container to outside the container. The container may have only one perforation, but it is preferred if it has at least 10 perforations in total, more preferably at least 50 or 100 perforations in total. Typically, the minimum dimension of any perforation is 0.25mm or more and the maximum dimension is no greater than 2mm or 1.5mm. Preferably, the minimum dimension of a perforation is 0.5mm or more. Also preferably, the maximum dimension of any perforation no greater than 1.2mm. More preferably, the maximum dimension is no greater than 1mm, still more preferably, no greater than 0.8mm. It will be appreciated that the precise minimum and maximum dimensions of the perforations will be determined by the ease of making the perforations, the intended thickness of the tissue slices to be retained in the container, the viscosity of the solutions to be used in the method and the desired speed of tissue draining; clearly, the maximum dimension of the perforation should not be so large so as to allow the tissue slice to pass through it, and the minimum dimension should not be so small that fluid cannot easily, or quickly, pass through it into or out of the container.

For example, when the tissue section being stained is 20 micron in thickness, the maximum dimension of the perforation may be about 1mm. When the tissue section being stained is 15 micron in thickness, the maximum dimension of the perforation may be about 0.8mm.

It will be appreciated that the maximum dimension of perforation which is suitable for a given size of tissue section may be determined by straightforward and routine experimentation.

The perforations in the container may be distributed anywhere in the container. The perforations may be distributed evenly in the container, and preferably the perforations are present at a density of more than 1 perforation per cm^"2, more preferably at a density of more than 2, 5, 10, 20 or 30 perforations per cm^" . It is preferred, however, if the perforations are located in a specific region of the container, and more preferably, the perforations are located close to the base of the container. For example, where the container is a cylinder, the perforations may be arranged in a circular fashion around the base of the cylinder. Each circular arrangement or line may have any number of perforations. Preferably a circular line of perforations has at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 or 10 perforations in it. It is preferred that where the perforations are arranged circularly around the base of the container, that there is more than one circular line of perforations, preferably at least 2, 3, 4, 5, 6, 7, or 8 or more circular lines of perforations. More preferably, there are 9 or 10 lines of perforations. Hence, for example, a container that has 8 circular lines of perforations wherein each line has 10 perforations, will have a total of 80 perforations. It is preferred if the distanced between any two perforations is at least 0.5mm, more preferably at least 1mm. Hence, one circular line of perforations may be separated from another line of perforations by 1mm.

Typically, the perforations in the container are sufficiently numerous and sufficiently large in diameter such that fluids or liquids may readily enter or exit the container via the perforations, whilst any tissue section within the container cannot exit the container via the perforations. By "readily", we mean that the container could be completely filled with fluid or liquid within 10 seconds, 5 seconds, 3 seconds or 1 second by means of fluid or liquid entering the container through the perforations alone (and not entering via the opening).

In a preferred embodiment, the container is a cylinder with perforations around the base as described above, and also has at least one perforation in the base of the container. Preferably, there are at least 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 50 or 60 or more perforations in the base.

An advantage of at least some perforations being located at and/or near the base of the container is that it allows a solution in the vessel to enter the container in step (iii) even when the amount of solution in the vessel is relatively small. A further advantage of the perforations being located at and/or near the base of the container is that it allows the solution to drain back into the vessel effectively (ie, without leaving a substantial amount of the solution in the container) as the container is removed from the vessel in step (v). Typically, the volume of solutions used in immunohistochemistry is relatively small, due to the high cost or scarceness of the antibodies or other solutions.

The opening of the container may be any suitable dimension. The opening of the container may be distinguished from a perforation in terms of its ability to allow a tissue section to pass through it. In other words, the opening of the container is one which can allow a tissue section to pass through it, whereas a perforation in that same container does not allow the same tissue section to pass through it. Typically, the opening is located at the top of the container, whereas the perforations are located towards the bottom of the container. Preferably, the opening has the at least same dimensions as the narrowest horizontal cross section of the container, more preferably the opening is wider than the narrowest horizontal cross section of the container. Hence, the shape of the container includes a flared cylinder or a frastroconical shape. In this case it is preferred that the open end is the widest end. The advantage of the open end having a larger cross sectional area than that of the rest of the container is that it may facilitate the insertion of the tissue sections into and removal out of the container. Furthermore, when such a container is used in conjunction with a vessel which is similarly shaped (ie, the opening of the vessel is larger than the cross section of the rest of the vessel such when the container is within the vessel, the walls of the vessel and container are closely associated with each other as defined above; for example, both the container and vessel are frustroconical), the addition of different solutions to the vessel using, for example, a pipette may be facilitated. Hence, the preferred dimension of the opening of the container is one which readily allows insertion or removal of tissue sections.

The container may be any convenient size. Preferably, the container is less than 100mm in width or diameter or depth, and greater than 7.5mm in width or diameter or depth. More preferably, the depth of the container is at least 15mm, 20mm, 30mm, 40mm or 50mm. Also preferably, the width or diameter of the container is at least 10mm, 15mm, 20mm or 25mm.

In a preferred embodiment, the container used in the method of the first aspect of the invention is one with at least one opening wherein the container

(i) is at least 7.5mm in width or diameter and at least 20mm in depth but less than 50mm in width or diameter and less than 100mm in depth; (ii) has sides which are straight in at least one plane; (iii) has at least ten perforations wherein the perforations in the container have a maximum dimension of 1.2mm; and is formed from plastic or glass which is no more than 1.5 mm in thickness.

An example of a container according to this aspect is shown in Figures 1 and 2. The vessel may be any suitable vessel that is capable of receiving the container. Preferably, the vessel has a depth that is less than the depth of the container, so that the container may be easily removed from the vessel by holding the top of the container. Alternatively or in addition, the container may have a handle region which extends beyond the vessel, so that the container can be held. The vessel may be made from any suitable material, such as glass or plastic. Preferably, the vessel is made from a material which does not chemically react with any solution or reagent to be used in the method, and does not adsorb biological molecules such as polypeptides onto its surface. Vessels which are suitable for use in the present method are available from most laboratory equipment suppliers such as Merck (Merck Ltd, Merck House, Poole, Dorset BH15 1TD; Product No. 275/0405/04).

Also preferably, the vessel is not substantially larger than the container, and more preferably, is the same or a similar shape. More preferably, the vessel is cylindrical or slightly frustroconical (for example, like a stacking beaker or cup) and still more preferably, the vessel has a flat base. The advantage of the container and vessel having the same or similar shape and not differing substantially in size is that when the container is placed in the vessel in step (ii), the walls of the container and vessel are closely associated. This close association means that the solution in the vessel will enter the container more easily in step (iii) and allow a smaller volume of solution to be used. The ability to use a minimal volume of solution is important where the solution contains, for example, antibodies which may not be readily available, or may be expensive.

By "closely associated" we mean that the maximum distance between a wall of the vessel and a wall of the container, when the container is placed in the vessel, is less than 50mm, preferably less than 25mm, 20mm, 15mm, 10mm or 5mm.

For example, where the container is 60mm in depth and has a diameter of 21mm, the vessel may usefully be 50mm in depth and have a diameter of 25mm. Conveniently, the vessel walls are at least 1mm thick. However, thickness of the vessel walls are not important in the same was as they may be for the container, since the vessel walls do not affect the movement of fluids.

In another embodiment, the container used in the method of the first aspect further comprises a cap that fits and can be secured onto the container.

By "cap" we mean a lid or covering device. The cap may be any convenient cap which does not impede the function of the container. For example, it is preferred if the cap is one whose shape and size permits the container to be placed inside the vessel of step (iii) so that the solution in the vessel inters the container via the perforations in the container. The cap may be made of any suitable material. The cap may be made of the same material as the container, as described above, or may be made from a different material. For example, the cap may be made from a flexible material, such as one which facilitates its securing onto, or removal from, the container. The cap may, or may not, be perforated. Where the cap is perforated, the perforation may be any convenient size. If the cap is perforated, it is preferred if the maximum dimension of any perforations is no greater than the maximum dimension of any perforation in the container, so that a tissue section which is retained by the perforations in the container is also retained by any perforations in the cap. The advantage of having perforations in the cap is that it may help easy displacement of air from the container as fluid enters it from the vessel, and may help prevent the generation of any bubbles in the fluid as it enters the container from the vessel.

The cap may or may not comprise a lip on the outer surface or along the outer edge. Preferably, the lip is one that can provide a structure against which pressure may be applied in order to more easily remove the cap from the container. An example of a suitable cap (comprising a lip) is shown in Figure 3.

By "secured" we include the meaning that the cap is attached to the container in such a way that it will not detach without some form of intervention. Means of securing caps to containers are well known in the art, and include threaded engagement whereby the cap and container have complementary threaded portions for threaded engagement one with the other.

By "fits" we include the meaning that when the cap is secured onto the container, there are no gaps between the container and the cap which allow a fluid such as water or a solution to pass through. In other words, when the cap is secured on the container, a fluid cannot leave or enter the container via the opening in the container.

The cap may be made from any suitable material; preferred materials are as those described above in relation to the container.

In one embodiment, the method further comprises the initial step of preparing the tissue section by sectioning and fixing a tissue. Methods of sectioning and fixing are discussed above. The method may further include a step of embedding the tissue prior to the steps of sectioning and fixing. In a further embodiment, the method further comprises step (vi) whereby steps (iii) to (v) are repeated using any second solution. By "second solution" we include any additional solution such as a second or third solution, a fourth, fifth or sixth and so on.

Hence, the method may involve repeating steps (iii) to (v) several times, either exposing the tissue section to the same solution, such as is useful when washing a tissue section or exposing the tissue section to more than one type of solution, such as to different stains or antibodies, or adding conjugates to antibodies for example. Typically, tissue staining involves incubating the fixed tissue section with phosphate buffers at physiological pH to wash the section, then hydrogen peroxide for deactivating any peroxidase enzymes, followed by blocking with serum/albumin/milk, exposing to a primary antibody of interest followed by a secondary biotinylated antibody. A biotinylated avidin biotin complex is ultimately formed which can be visualized after addition of diaminobenzidine and hydrogen peroxide. .

A second aspect of the invention provides a container with at least one opening wherein the container

(i) is at least 7.5mm in width or diameter and at least 20mm in depth but less than 50mm in width or diameter and less than 100mm in depth; (ii) has sides which are straight in at least one plane; (iii) has at least ten perforations wherein the perforations in the container have a maximum dimension of 1.2mm; and is formed from plastic or glass which is no more than 1.5 mm in thickness. The container is one which is useful in the first aspect of the invention, and preferably dimensions and features are as described in relation to the first aspect of the invention.

An example of a container according to the invention is shown in Figures 1 and 2.

A third aspect of the invention provides a cap which fits and can be secured onto a container as defined in the first or second aspect of the invention in combination with said container.

A "cap" is as defined above in relation to the first aspect of the invention.

By "secured" and "fits" we include the meanings as defined above.

The cap may, or may not, have one or more perforations. Where the cap has at least one perforation, it is preferred if the perforation or perforations are as described above in respect of the container. The perforation or perforations may be any convenient size or sizes, such as about 1mm, 2mm, 3mm, 5mm, 7mm or more in diameter. Advantageously, the perforations have a dimension which is greater than the largest perforation in the container.

By "in combination" we include the meaning that the cap and container are provided together, preferably in contact with each other, and more preferably the cap is attached to the container as described above.

In one embodiment, the cap and container of the invention, or useful in the invention, are parts of the same article, forming a one-piece article; for example, they are joined by a flexible hinge region, as is well known to those skilled in the art.

Preferred forms of the container are as described above.

A fourth aspect of the invention provides a use of a container as defined in the first or second aspect or a cap in combination with a container as defined in the third aspect in a method of tissue section staining.

The tissue section staining may be any type of tissue section staining. Preferably, the staining is immunohistochemical staining.

It will be appreciated that the use may be in any stage of a tissue section staining method, including the preparatory stage. Hence, the invention provides a use of a container as defined in the first or second aspect or a cap in combination with a container as defined in the third aspect in a method of general cleaning, dehydrating, de-fattening or de-waxing a tissue section. Typically, tissue sections are subjected to cleaning, dehydrating and de- fattening in the preparation for the addition of one or more stains.

A fifth aspect of the invention provides a method of making a container as defined in the second aspect of the invention comprising forming a container made of glass or plastic which is at least 7.5mm in width or diameter and at least 20mm in depth but less than 50mm in width or diameter and less than 100mm in depth and has sides which are straight in at least one plane such that the container has at least ten perforations which perforations have a maximum diameter of 1.2mm.

The perforations may be made by any convenient means. Typically, the perforations are made using a fine drill. Alternatively, the perforations may be introduced into the container during the production of the container itself, such as during the moulding of the container. Plastic and glass moulding techniques are well known in the art.

A sixth aspect of the invention provides a kit comprising a container as defined in the first or second aspects of the invention and a vessel which has a diameter or width which is larger than the diameter or width of the container.

The container is as described and preferred above. Suitable and preferred vessels are as described above.

According to a preferred embodiment, the kit further comprises a cap which fits and can be secured onto said container. Preferred caps are as described above.

A seventh aspect of the invention provides a kit comprising a container as defined in the second aspect of the invention and a cap which fits the container and can be secured onto said container.

According to a preferred embodiment of this aspect of the invention, the kit further comprises a vessel. Suitable vessels are as described above.

Where the kit of the invention comprises a cap, the preferred form of the cap is as described above. Hence, the cap may or may not have one or more perforations. Preferably, any perforations in the cap have a maximum dimension of 1.0mm. Conveniently, the kit of the invention may further comprise a fixing reagent. Fixing reagents are well known in the art of tissue fixing, and include formalin, paraformaldehyde and glutaraldehyde, amongst others.

The kit of the invention may also, or instead of, further comprise at least one staining solution. A staining solution may be any solution which is useful in a method of tissue section staining, including phosphate buffers, blocking solutions such as albumin or serum, primary antibodies, biotinylated secondary antibodies, and avidin-biotin complex.

The invention will now be described in more detail with the aid of the following Figures and Example. Figure 1

Photograph and line drawing representing a perspective view of an example of a container. In this case, the container is made from polymethyl methacrylate (Perspex™) and the perforations are 1mm in diameter.

Figure 2

(A) Photograph and line drawing representing an end view of the container shown in Figure 1, showing perforations of 1mm diameter in the base

(B) Photograph and line drawing representing a side view of the container shown in Figure 1.

Figure 3 Photograph and line drawing representing a perspective view of an example of a cap. The perforation in the cap is about 4.5mm.

Figure 4

Photograph and line drawing representing a perspective view of an example of a vessel useful in the invention. This vessel is made from glass. Figure 5

(A) Photograph and line drawing representing a perspective view of an example of the container shown in Figures 1 and 2 placed in the vessel shown in Figure 4 with a cap (as shown in Figure 3) fitted onto the container.

(B) Photograph and line drawing representing a side view of the same ensemble of container, cap and vessel shown in (A)

Example 1

Tissue sections greater than 20 microns in thickness are cut, pre-fixed, and placed into a container as shown in Figure 1. This container is made from Perspex™. A cap, such as the plastic cap shown in Figure 3, is fitted over the opening of the container. A staining solution, such as an antibody solution, is added to a vessel such as the glass vessel shown in Figure 4, and the container is placed into the vessel. The antibody solution enters the container via the perforations near the base, and in the base of the container. The container remains in the vessel to incubate the tissue sections in the antibody solution for as long as is required. Once the incubation step is completed, the container is raised from the vessel, and the antibody solution drains away as the container is raised.

Claims

1. A method of staining a tissue section comprising the steps of

(i) Providing a fixed tissue section;

(ii) Placing the tissue section of step (i) into a container with at least one opening and at least one perforation wherein the container is at least 0.75cm in width or diameter or depth but less than 10cm in width or diameter or depth wherein the at least one perforation has a maximum dimension of 2mm;

(iii) Placing the container comprising the tissue section in a vessel containing a first solution such that the solution in the vessel enters the container via the perforations in the container; (iv) Incubating the tissue section in the first solution; and (v) Removing from the container at least some of the solution via the perforations in the container.

2. A method according to Claim 1 further comprising the initial step of preparing the tissue section by sectioning and fixing a tissue.

3. A method according to Claim 1 or 2 further comprising step (vi) whereby steps (iii) to (v) are repeated using any second solution.

4. A method according to Claims 1 to 3 wherein the container has sides which are straight in at least one plane.

5. A method according to Claims 1 to 4 wherein the container has a flat base.

6. A method according to any one of Claims 1 to 5 wherein the container is made of plastic or glass.

7. A container according to Claim 6 wherein the plastic is polymethyl methacrylate.

8. A method according to any one of Claims 1 to 7 wherein the container has more than one perforation and they are present at a density of more than 1 perforation per cm^" .

9. A method according to Claim 8 wherein the perforations are present in a density of more than 10 holes per cm^" .

10. A method according to any one of Claims 1 to 9 wherein the total number of perforations in the container is more than 10.

11. A method according to any one of Claims 1 to 10 wherein the maximum dimension of a perforation in the container is 1mm.

12. A method according to Claim 11 wherein the maximum dimension is 0.8mm.

13. A method according to any one of Claims 1 to 12 wherein the material forming the container is no more than 1.5mm thick.

14. A method according to Claim 13 wherein the material is no more than 1.0mm thick.

15. A method according to any one of Claims 1 to 14 wherein the depth of the container is at least 30mm.

16. A method according to Claim 15 wherein the depth is at least 40mm.

17. A method according to any one of Claims 1 to 16 wherein the minimum width or diameter of the container is 10mm.

18. A method according to Claim 17 wherein the minimum width or diameter is 20mm.

19. A method according to any one of the previous claims wherein the container is cylindrical.

20. A method according to any one of the preceding claims wherein the container is used in combination with a cap that fits and can be secured onto said container.

21. A method according to Claim 20 wherein the tissue is sectioned into slices no thicker than 20 microns.

22. A method according to Claim 21 wherein the perforations in the container have a maximum dimension of 1mm or less.

23. A method according to any one of Claims 1 to 20 wherein the tissue is sectioned into slices no thick than 15 microns.

24. A method according to Claim 23 wherein the perforations in the container have a maximum dimension of 0.8mm or less.

25. A container with at least one opening wherein the container (i) is at least 7.5mm in width or diameter and at least 20mm in depth but less than 50mm in width or diameter and less than 100mm in depth; (ii) has sides which are straight in at least one plane; (iii) has at least ten perforations wherein the perforations in the container have a maximum dimension of 1.2mm; and (iv) is formed from plastic or glass which is no more than 1.5 mm in thickness.

26. A container according to Claim 25 which is cylindrical.

27. A container according to Claim 25 or 26 wherein the plastic is polymethyl methacrylate.

28. A container according to any one of Claims 25 to 27 wherein the perforations have a maximum dimension of 1.0mm.

29. A cap which fits and can be secured onto a container as defined in any one of Claims 25 to 28 in combination with said container.

30. A cap according to Claim 29 which is perforated with at least one perforation.

31. A cap according to Claim 30 wherein the maximum dimension of the perforation is 1.2mm.

32. Use of a container as defined in any one of Claims 1 to 28 or a cap in combination with a container as defined in any one of Claims 29 to 31 in a method of tissue section staining.

33. Use according to Claim 32 wherein the tissue section staining is immunohistochemistry.

34. Use according to Claim 32 wherein the use is in any one or more of a method of general cleaning, dehydration, de-fattening or de-waxing prior to addition of a stain.

35. A method of making a container as defined in any one of Claims 25 to 28 comprising forming a container made of glass or plastic which is at least 7.5mm in width or diameter and at least 20mm in depth but less than 50mm in width or diameter and less than 100mm in depth and has sides which are straight in at least one plane such that the container has at least ten perforations which perforations have a maximum diameter of 1.2mm.

36. A method according to Claim 35 wherein the perforations are made using a fine drill.

37. A kit comprising a container as defined in any one of Claims 1 to 28 and a vessel which has a diameter or width which is larger than the diameter or width of the container.

38. A kit according to Claim 37 further comprising a cap that fits and can be secured onto said container.

39. A kit comprising a container according to any one of Claims 25 to 28 and a cap which fits the container and can be secured onto said container.

40. A kit according to Claim 39 further comprising a vessel which has a larger diameter or width than the diameter or width of the container.

41. A kit according to any one of Claims 37 to 40 wherein the cap has at least one perforation wherein the maximum dimension of the at least one perforation is 1mm.

42. A kit according to any one of Claims 37, 38 or 40 wherein the vessel is less deep than the container.

43. A kit according to any one of Claims 37 to 42 further comprising a fixing reagent.

44. A kit according to any one of Claims 37 to 43 further comprising at least one staining solution.