WO2006039396A2 - Non-embedded tissue microarray technology for protein and nucleic acid analyses - Google Patents

Abstract

The current invention relates to methods, compositions, and/or devices for preparing a tissue sample comprising blocks. Methods of the invention generally comprise: obtaining a tissue sample recipient block comprising a matrix material and a first cryostat protection component, said block having at least one array cavity disposed therein; obtaining at least one tissue sample that is unfixed or has been fixed in a solution comprising a fixative and a second cryostat protection component; and placing the sample in an array cavity in the block. Sections of blocks obtained using these methods, compositions, and/or devices, and uses of such sections as tissue microarrays are also contemplated.

Description

DESCRIPTION

NON-EMBEDDED TISSUE MICROARRAY TECHNOLOGY FOR PROTEIN AJXD NUCLEIC ACID ANALYSES

BACKGROUND OF THE INVENTION This application claims the benefit of the filing date of U.S. provisional patent application Serial No. 60/615,248, filed October 1, 2004, the entire contents of which are hereby incorporated by reference.

1. Field of the Invention The present invention relates to the field of biomedical science and/or molecular biology. More specifically, the invention relates to methods for making sections of tissue samples, including but not limited to tissue microarrays.

2. Description of Related Art Tissue microarray (TMA) techniques involve arraying multiple cylindrical tissue cores from different tissue specimens on a single glass slide. TMA is a high throughput molecular biology technique that enables the rapid validation of the detection of mRNA or protein markers of different human tissue samples in one experiment. This technique is widely used for a variety of human diseases. Altered expression patterns of the genes and proteins in the TMA samples can be correlated to the mechanisms of human pathologies and provide valuable information for clinical diagnostics and therapies.

One of the main uses for TMAs is for oncology studies. Often, hundreds of different tumor samples at different stages and grades are used to prepare the TMAs (Schrami et al, 1999; Moch et al, 1999; Bubendorf et al, 1999a; Bubendorf et al, 1999b). With TMAs of this type, a large group of tumors can be expeditiously screened and carefully compared for the presence of novel markers (Andersen et al, 2002). For example, a tumor progression array has been used to demonstrate that the expression of a novel protein, EZH2, correlates with the aggressiveness of prostate cancer (Varambally et al, 2002). Similar studies using TMAs reveal the relationship between the her-2 gene and breast cancer (Simon et al, 2001). TMAs are also used for clinical immuno- histochemical diagnostics (Hsu et al, 2002) and for functional target validation (Mousses et al, 2002). Based on these facts, it appears likely that the investigation of pathogenesis and progression of human diseases will lead to the increased use of TMAs. Advances in TMA technology will likely lead to an increase in its utility as a research tool.

A high quality TMA should have the following four features: well protected nucleic acids, intact proteins, clear cell morphology, and user-friendliness. However, there are currently challenges in producing TMAs that meet these criteria in an efficient, replicable, consistent manner that will allow for the preparation of a large number of TMAs. Further, many of the most important TMA applications of the present and future will require the analysis of RNA, which is an extremely labile molecule as compared to DNA. Therefore, techniques that allow for the preservation of RNA in TMAs are needed.

A variety of techniques have been developed to make TMAs (reviewed by Shergill et al., 2004). In 1986, Battifora introduced a "sausage" block method, in which multiple 1 mm thick "rods" of tissue samples were wrapped in a sheet of small intestine. The wrapped tissue was then embedded in a paraffin block from which sections were cut for studies. Later on, Basttifora et al. (1990) further improved upon their "sausage- block" technique creating a "checkerboard" configuration. Although this technique had the significant advantage of simultaneously examining multiple tissue specimens under identical conditions, it also had significant disadvantages including that only a few dozen individual specimens could be arranged per array and the sizes and configurations of each individual tissue sample were not identical.

Attempting to overcome the limitations of the "sausage" technique, Kononen et al. (1998) used a thin-well stainless steel tube to take cylindrical tissue biopsies from paraffin-embedded tissues and then, with the same tube, transferred tissue cores into defined array coordinates in a recipient paraffin block. Several hundred array sections can be cut from the block. This paraffin-embedded array technique allows arraying up to 1000 cylindrical tissue cores on a TMA, in which the sizes and configurations of the individual cores are identical. Kononen' s method (Kononen, 1998) generally produces reproducible protein expression patterns but the RNA qualities of such samples exhibit obvious variations. KNA is very susceptible to degradation (Sambrook et al., 1989) and is readily hydrolyzed when exposed to conditions of high pH, metal cations, high temperatures, and contaminating ribonucleases (RNase). Most RNA molecules in the paraffin-based TMA were degraded.

Although quick fixation of tissue samples with fixatives such as formalin and paraformaldehyde results in the cross-linking between RNA and protein (Masuda et al, 1999), fixatives themselves might not directly cause degradation of RNA molecules. In contrast, fixatives can inhibit RNase activities in cells to prevent RNase-induced RNA degradation. Fixative-induced cross-linking between RNA and protein affects the hybridization efficiency for RNA detection and affects integrity of RNA. RNA degradation might occur during the paraffin-embedding and deparaffinization processes. This was suggested by the fact that changing the fixatives from cross-linking reagents, like formalin, to non-cross-linking reagents, like ethanol, did not significantly improve the quality of RNA in tissue. It has been shown that paraffin embedding of tissues fixed by non cross-linking fixatives such as ethanol does not prevent RNA degradation (Goldsworthy et al. , 1999).

Although a new paraffm-based method for the preparation of TMAs has been recently suggested (Dan et al, 2004), it does not provide a solution to the RNA degradation problem. The only improvement in Dan's method is precise localization and isolation of representative regions in the donor blocks by removing donor tissue under a modified microscope. Protection of RNA molecules from degradation is one of the very important parameters to be considered when making a TMA.

To further improve the quality of both proteins and RNAs in TMA samples, a new OCT-embedded frozen tissue-based TMA technology has recently been developed by Slamon (U.S. Patent 6,696,271 B2). The term "OCT" refers to the compounds utilized to rapidly embed biological materials for sectioning. The OCT medium is sold by Tissue Tek under the name optimal cutting temperature compound (product code 4583) and the medium sold by Instrumeducs Inc, under the name "Cryo-Gel" (Cat#ICG-12). In place of paraffin, they embedded non-fixed tissue samples in OCT compound in dry ice and then used these embedded frozen tissue samples as donor samples to array into a recipient OCT block. Sections of the array block are cut using a cryostat microtome. The TMAs created from this technology have been successfully used for RNA in situ hybridization and immunocytochemistry studies (Fejzo et al, 2001). Although this method circumvents the RNA degradation problem observed in paraffin-based TMA, some technical problems still exist. There are significant drawbacks to the OCT-embedded frozen tissue-based TMA technology. To prevent melting of the OCT compound, all the experiments must be performed on dry ice, which is very inconvenient. Further, coring array samples from frozen tissues embedded in OCT is difficult because the tissue samples become solid and hard when frozen in dry ice, and the needles are easily bent. It is also technically difficult to drill array cavities inside OCT blocks frozen in dry ice. Expensive instruments such as TNA arrayer (Beecher Instruments, MD) are required, which significantly compromises the ability of most academic and clinical laboratories to prepare TMAs in a low throughput setting, hi addition, the tissue samples tend to adhere to the inside of the coring tube, due to the temperature difference between the coring tube and cored tissue.

Another drawback to the OCT-based TMA technology is that only a few samples can be embedded on a single array because the OCT compound may bend and crack when samples are placed less than 1 mm apart.

Furthermore, after arraying cored tissues into an OCT block, a re-melting procedure is required to keep the tissue cores inside the OCT block during sectioning. This re-melting procedure can damage the tissue in the cores and result in alterations of cell morphology in the tissue cores.

As mentioned above, a high quality TMA will typically have the following features: well-protected cell morphology, high quality of proteins and nucleic acids, good cell morphology, and user-friendliness. Based on the above analyses, it is unlikely that neither of the two previously mentioned techniques can meet each of these criteria.

Consequently there is a need in the art to identify additional methods enabling the optimal preservation of not only the biological molecules, such as proteins and RNA but also preserve cell morphology. The present irrvention meets these specifications by providing a method that circumvents the problems associated with present available technologies.

SUMMARY OF THE INVENTION

The present invention provides a new technique to prepare sections, including but not limited to TMAs. In some particular embodiments, the invention disclosed herein improves upon existing technology by arraying fixed specimens into a recipient block that comprises a modified gel. These methods can include the steps of preparing an array recipient block, coring fixed frozen tissue samples, arraying tissue samples into an array block, and sectioning the array block. In this invention, the block contains multiple pre-made arrayed cavities and the tissue is either embedded or not embedded, prior to arraying. The quality of the sections from array samples can be evaluated according to the appropriate methodologies used to analyze cell morphology and specific gene expression at the RNA and protein levels. TMAs made by this new technique present higli quality of both RNAs and proteins, and excellent cell and tissue morphologies. These methods may further include manufacturing instruments for making an array block.

The embodiments of the present invention present a number of advantages over conventional paraffin-based technology. The present method overcome problems associated with RNA quality, which are commonly observed in the paraffin-based methods. The overall time to make the array sample is much shorter and the cost is significantly cut down. No xylene, a toxic reagent, need be used in the present invention. The invention also has a number of advantages over the OCT-based methods. For example, the issues of fracturing during sectioning are overcome. Other advantages are that both embedded and non-embedded samples can be used and that the array block can be made at either room temperature or low temperature. In embedded tissue materials, one section must be cut and stained by H&E staining. Stained sections are overlaid on top of the original tissue block to find the right place to core. In the non-embedded samples, tissue material can be cored directly from samples and placed into the array block. Double punches are also possible.

In broad embodiments, the current invention relates to methods of preparing a tissue sample comprising block, the method comprising: obtaining a tissue sample recipient block comprising a matrix material and a first cryostat protection component, said block having at least one array cavity disposed therein; obtaining at least one tissue sample that is unfixed or has been fixed in a solution comprising a fixative and a second cryostat protection component; and placing the sample in an array cavity in the block. In many embodiments, the block comprises a plurality of array cavities. For example, the block may comprise 2, 4, 5, 9, 10, 16, 20, 25, 30, 36, 40, 49, 50, 64, 75, 81, 96, 100, 121, 125, 144, 150, 169, 175, 196, 200, 225, 250, 300, 400, 500, 100O, any integer between these numbers, or more array cavities or any range of array cavities between any two of these numbers. In some specific embodiments, the block: may comprise 10 or more array cavities and/or 96 or more array cavities.

These methods may further comprise obtaining a plurality of samples that are unfixed or have been fixed in the solution and placing the plurality of samples individually in a plurality of array cavities in the block. The methods may also comprise acquiring unfixed multiple samples and arraying those samples individually In the recipient block. For example, the method may comprise placing at least 10 samples in at least 10 array cavities in the block and/or at least 96 samples in at least 96 array cavities in the block.

The matrix material can be any matrix material that is known in the art and that allows for the preparation of tissue sample blocks that will function in the methods and compositions of the invention. Those materials include agarose, gelatin, paraffin and others that will be understood by those of skill upon reading this specification. Lx some preferred embodiments, the matrix material is agarose. For example, the block may comprise 0.1% to 15% agarose. Any type of agarose known to those of ordinary skill in the art, such as low temperature and high resolution agarose, can be used. Ix more specific embodiments, the block comprises 0.3% to 12% agarose, and, for some presently preferred applications, the block comprises 0.5% to 10% agarose.

The tissue material can be any tissue sample from any species that alloΛvs for preparing TMA blocks. Examples include fresh tissue samples, frozen tissue samples, and tissue samples fixed by different kinds of fixatives. One of ordinary skill in the art would be familiar with use of various fixatives to fix tissue samples. Fresh or frozen tissue samples can also be used directly to preparation after being embedded in gelatin or OCT. Embedded tissue samples can be cored immediately to array into recipient block to make an array block. Tissue samples can also be first pre-treated with a ciyostat protection components and then used for preparation of TMA blocks.

The cryostat protection components of the invention can provide several benefits in the context of the invention. Fresh or frozen biological material is typically difficult to manipulate without cracking or otherwise compromising the integrity of the tissue. Consequently, a skilled artisan would expect some frozen materials to crack: when exposed to the significant mechanical stress associated with the tissue. Microarray protocol. Such stresses include those associated with the coring of the frozen tissues as well as the stresses associated with cutting 4-10 micron sections from the Microarray block prior to their analysis. Cryostat protection facilitates tissue sectioning in a -20°C cryostat microtome. As a result, good cell and tissue morphologies will be present in the sections. In the context of the invention, the first cryostat protection component, which is comprised in the matrix, serves to make the block easily sectionable at a freezing or close to freezing temperature and facilitates the preparation of intact matrix sections. In the context of the invention, the second cryostat protection component serves to protect cellular morphology in samples fixed in the solution comprising the second cryostat protection component. The first and second cryostat protection components may be the same or different in a given embodiment of the invention, depending upon the needs for that particular embodiment. Any agent that results in the desired characteristics of the cryostat components of the invention may be used in this regard. For example, the first and/or second cryostat protection component can be, but is not limited to, a saccharide, glycerol, and alcohol. In some preferred embodiments, the cryostat protection component is a saccharide, for example sucrose. In some preferred embodiments, the saccharide is sucrose.

The first cryostat component may be comprised in the block in any suitable concentration. For example, between about 5% and 25%, between about 10% and 20%, and/or between about 10% and 15%. Some specific embodiments comprise about 13.5% of the first cryostat protection component. Some specific embodiments comprise between about 5% and 25% sucrose, between about 10% and 20% sucrose, between about 10% and 15% sucrose, and/or about 13.5% sucrose. In some other embodiments the block comprises an antimicrobial agent. The antimicrobial agents include fixatives, antibiotics, peptides, and other chemicals such as silvazine and dimethyl sulfoxide. For example, the microbial agent can be paraformaldehyde. For example, the block may comprise 0.01% to 0.1% paraformaldehyde or even more specifically 0.01% to 0.04% paraformaldehyde.

The fixative in the solution may be any form of fixative that is acceptable to function in the context of the invention. In some embodiments, the fixative is paraformaldehyde, formaldehyde, buffer formalin, glutaraldehyde, ethanol, or acetone. In some preferred embodiments, the fixative is paraformaldehyde. The fixative may be comprised in the solution in any suitable concentration. For example, between about 1% and 10% and/or between about 3% and 6%. Some specific embodiments comprise about 4% of the fixative. Some specific embodiments comprise between about 1% and 10% paraformaldehyde, between about 3% and 6% paraformaldehyde, and/or about 4% paraformaldehyde. The second cryostat protection component can be any component that is capable of accomplishing the cryostat goals of the invention by preserving sufficient sample morphology for a given application. In some embodiments, the second cryostat protection component is a saccharide, glycerol, alcohol, and so on. In some preferred embodiments, the second cryostat protection component is a saccharide, for example, but not limited to, sucrose. The second cryostat component should be comprised in the solution in an amount sufficient to accomplish the goals of the invention by preserving morphology. Some embodiments comprise between about 10 and 50% of the second cryostat protection agent, between about 15% and 40% of the second cryostat protection agent, between about 20% and 35% of the second cryostat protection agent, and/or about 30% of the second cryostat protection agent. Some preferred embodiments comprise between about 10% and 50% sucrose, between about 15% and 40% sucrose, between about 20% and 35% sucrose, and/or about 30% sucrose. In preferred embodiments, the solution preserves cellular morphology in a frozen sample or a fresh sample. In preferred embodiments the solution maintains RNA quality and/or quantity in the sample.

In some particular preferred embodiments, the matrix is agarose, the first cryostat protection component is sucrose, the fixative is paraformaldehyde, and the second cryostat protection component is sucrose.

The methods of the invention may further comprise sectioning the block to obtain a section. In specific embodiments, the sectioning results in the preparation of a TMA. In some embodiments, the sectioning comprises sectioning a 1 μm to 20 μm thick section comprising a portion of the sample. In more specific embodiments the section is 2 μm to 10 μm thick and/or about 4 μm thick. In most embodiments, the first cryostat protection component facilitates sectioning of the block to produce an intact section comprising a portion of the sample surrounded by matrix. An "intact" section is a section that is usable in a desired application, for example in a TMA. Typically, intact sections are substantially flat or capable of being made substantially flat, are substantially uncracked, and are substantially not folded. An additional value of the cryostat protection component is the fact that in some embodiments, the combination of matrix and the first cryostat protection component allow for easy manipulation of the section. In some embodiments, an in situ hybridization is further performed on the section. Other embodiments comprise performing immunohistochemistry on the section. Embodiments may comprise dehydrating the section and/or taking other preparative steps known to those of skill.

The methods of the invention may further comprise analyzing nucleic acid, for example RNA or DNA, in the section and/or isolating such nucleic acids from the section. The methods of the invention may also further comprise analyzing and/or isolating protein from the section. hi most embodiments of the invention, the embedded or non-embedded tissue sample is frozen prior to placement in the block. Freezing facilitates cutting or coring of the sample and placement of it in the block, hi some cases, the sample has been cut or cored out of a larger piece of frozen or unfrozen tissue. The temperature of the frozen tissue is below 4°C (and preferably less than about -15-2O⁰C). For example, the tissue samples may be frozen at -20°C or lower and cored with a sharp steel tube. Further, obtaining the first tissue sample may comprise obtaining a core of tissue that fits within one of the array cavities, hi most cases, the sample will be cut or cored at room temperature or at -10°C to 15°C from a larger frozen block of tissue. Cored tissue samples can then be arrayed into one of the array recipient blocks at room temperature or at low temperature such as -1O⁰C to -15⁰C. In some embodiments, the sample could be fixed or unfixed. The sample may also be defined as an archival sample, i.e., one that has been stored for some period of time prior to placement in the block. The sample may be obtained from a live or dead organism.

In some specific embodiments, the invention comprises making the block by placing liquid matrix in container; placing a device comprising a plurality of pins in the liquid matrix, and allowing the matrix to solidify.

One of the advantages of the invention is its ability to allow for sectioning protocols that preserve morphology and RNA. In this regard, the invention allows one to control the temperature at which the various steps are conducted, in such a manner that these goals can be achieved. For example, in some embodiments, fresh or frozen tissues samples can be immersed in fixatives containing 20-30% sucrose at a temperature of about 4°C. After sinking in the fixative solutions, tissue samples can be removed from fixatives and quickly frozen for 10-15 min in a -2O⁰C freezer or for 3-5 min in dry ice to let the tissue become slight hard to facilitate coring. Cored tissue samples can then be arrayed into the recipient block at room temperature or at about 4°C. The final array block can then wrapped to prevent the dehydration of tissue arrays during preservation and then kept in -80⁰C freezer to prevent the degradation of mRNAs and proteins. Of course, those of skill in the art will, in view of this specification, be able to make an of a number of changes in the temperature parameters above and still realize the benefits of the invention in the context of a given use.

Some embodiments of the invention relate to tissue sample recipient blocks comprising a matrix material and a cryostat protection component, said block having at least one array cavity disposed therein. These blocks may be further defined as a microarray recipient block. Tissue sample recipient blocks of the invention can comprise a plurality of array cavities as defined herein, for example, but not limited to, at least 10 array cavities or least 96 array cavities. In some embodiments, the matrix is agarose in a suitable percentage as defined above. The cryostat protection component in the block can be any such component in a suitable concentration as defined above, with sucrose being one presently preferred cryostat protection component. The tissue sample recipient block may further comprise an antimicrobial agent, for example, paraformaldehyde. Tissue sample recipient blocks of the invention may be further defined as comprised in a package. Such packaged blocks may be made in one location and transferred to another prior to use. In most cases, the package is further defined as a sealed package that prevents drying of the microarray recipient block. For example, the package can be a plastic box. The tissue array recipient block may be further defined as comprised in a kit.

Other embodiments of the invention relate to fixative solutions comprising a fixative and a cryostat protection component, as discussed above. In some preferred embodiments, the fixative is paraformaldehyde in any suitable concentration. In some preferred embodiments, the cryostat protection component in the solution can be a saccharide, for example, but not limited to, sucrose, in any suitable concentration as defined above. Solutions of the invention will, in most embodiments, preserve cellular morphology in a frozen or fresh sample fixed in the solution. Additionally, solutions of the invention will, in some embodiments, preserve RNA quality and/or quantity in a sample fixed in the solution. The solutions of the invention may be comprised in a suitable container and provided as a stand-alone product or as part of a kit.

In other embodiments, the invention relates to sections comprising, as defined above, a matrix comprising a matrix material and a first cryostat protection component; and at least one tissue sample that has been fixed in a solution comprising a fixative and a second cryostat protection component disposed within said matrix. Such sections may be further defined as TMAs. The section may comprise a plurality of tissue samples disposed in said matrix. In other embodiments, the invention relates to kits. For example, such kits may comprise, as defined above, a microarray recipient block comprising a matrix material and a first cryostat protection component, said block having a plurality of array cavities disposed therein; and a fixative solution comprising a fixative and a second cryostat protection component. Kits of the invention may comprise a punch for preparing a core tissue sample. The kits may comprise one or more reagent for making a matrix with a first cryostat protection component and/or a fixative solution comprising a fixative and a second cryostat protection component. Kits of the invention may comprise one or more components of a system for making a microarray recipient block, as defined below. A related embodiment of the invention includes a method for preparing an instrument system to make a tissue sample recipient block comprising the steps of: manufacturing a set of instruments comprising a box and a stamp with multiple pins on it; melting or dissolving array matrix material; pouring liquid matrix into the box; placing the stamp in the box; and removing the stamp after matrix solidification. Some specific embodiments of systems of the invention are defined as adapted for forming a tissue sample recipient block comprising a matrix material, said block having at least one array cavity disposed therein, the system comprising at least an array cavity forming device (or stamp) comprising at least one member configured to form an array cavity in the matrix material operably disposed on a support. Such systems may be further defined as systems for preparing a microarray recipient block. In some embodiments, the at least one member is further defined as a steel pin or a pin of other suitable material. The pin, in specific embodiments, may be of a diameter of from 0.6 to 1.0 mm. The pin may be 1.0 to 2.5 cm long, or any other suitable length, with some preferred pins being 1.5 to 2.0 cm long. A plurality of members may be operably arrayed on the support, for example at least 10 members and/or least 96 members. The members may be arrayed on the support in any suitable pattern. For example, the members may be arrayed on a 1.0 to 4.0 cm by 1.0 to 4.0 cm region of the support, a 1.5 to 2.0 cm by 1.5 to 2.0 cm region of the support, and/or a 1.5 by 2.0 cm region of the support. The system may further comprise a handle operably coupled to the support. In some cases, the handle is coupled to the support on a side opposite the at least one member. Additionally, the system may comprise a mold into which liquid matrix material and the array cavity forming device can be introduced during use. In many embodiments, the mold is plastic, although it can be of any suitable material including but not limited to metal or glass. In some particular embodiments, the mold is polycarbonate. The mold can be any suitable size to receive the array cavity forming device, for example from 1.0 to 5.0 cm by 1.0 to 5.0 cm. A currently depth of the size is about 1.0 cm. In some embodiments, the mold is a box of 3.0 cm by 4.0 cm by 1 cm. The mold may have a lid, which may have defined therein a hole through which a handle of the array cavities forming device will fit during use. The mold may also have, in its interior bottom, a series of recesses adapted to receive and position the members during use.

AU terms of art, notations and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to

Λvhich this invention pertains. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology described in Sambrook et al. (1989).

The term "tissue samples" is used herein according to its broadest meaning and refers to the wide variety of biological materials that can be analyzed in TMAs, including tissues from specific organs such as brain, kidney, liver, heart, bone, prostate, and other tissues. Such materials also include in vivo and in vitro cellular materials such as cancer cell lines.

A "plurality," as used herein, denotes "two or more." It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

Other objects, features and advantages of the present invention will become apparent to those skilled in the art following detailed description. It is to be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration and not limitation. Many changes and modifications within the scope of the present invention may be made without departing from the spirit thereof, and the invention includes all such modification.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein:

FIG. IA, FIG. IB, FIG. 1C, FIG. ID. Illustrations show the instruments that are used to make tissue Microarray block matrix. FIG. IA and FIG. IB are lateral views of the instrument. FIG. 1C and FIG. ID are dorsal views of the instrument. The instrument comprises two parts: the plastic box that is used to hold dissolved matrix (indicated by short arrows, 50, in FIG. IA and FIG. IB) and the steel stamp mold which contains multiple steel pins (indicated by long arrow, 30, in FIG. IB). After pouring the dissolved matrix into the plastic box, the steel mold is placed into the box. After the dissolved matrix solidifies, the steel mold is removed. The spaces occupied by the small pins become long round holes. Those array cavities are used to hold cored tissue samples.

FIG. 2. The matrix used for making human TMA recipient blocks. A numerous long round cavities are pre-made by the instrument described in FIG. 1. This block is ready-to-use to make a TMA block.

FIG. 3. Illustration shows the matrix after sectioning. All 96 small cores may be used in this matrix. No folding or curing occurs with the matrix. FIG. 4. Microarray method and H&E staining. A total of 96 1.0 mm samples from human brain and other organs spaced 1.0 mm apart were placed in agarose gel based matrix. After the array is completed, the block is put into a cryostat microtome for sectioning. A 14-micron section of the block is stained with H&E staining to show the overall morphology of tissue array.

FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D, FIG. 5E. Cresyl violet and H&E stained (B-E) TMA sections. FIG. 5A - AU the array cores were well aligned in the slide as revealed by cresyl violet staining (upper panel) and H&E staining (low panel). Each core has similar size and regular round shape. FIG. 5B - A stained 1.5 mm tissue core made from fresh fixed brain cerebellum. The overall cerebellum structure is well defined. The edge of the tissue core is sharp and the granule cell layer is clear present inside the brain (arrow). FIG. 5C - The inner structure of frozen pancreas cancer tissue observed under 2OX magnification. The structure in this frozen tissue is well preserved. No debris or freezing- or thawing-induced morphological damage was observed. FIG. 5D - Detailed structure of cerebellum Purkinje cells (arrow) and granule cells in fresh fixed cerebellum. No empty holes and separation of cells are present inside the section. The cell membranes of the Purkinje cells are clear and intact. FIG. 5E - Frozen lung cancer sample demonstrating the typical nuclear profiles of cancer cells (arrow). The nuclei are large, with irregular shapes. The edge of cancer cells is clear and no empty holes and debris are seen in the section.

FIG 6A, FIG. 6B, FIG. 6C, FIG. 6D. Non-isotopic RNA in situ hybridization is performed using fluorescein-labeled vasoactive intestinal polypeptide (FIG. 6A, FIG. 6B) and human GAPDH antisense RNA probe (FIG. 6C, FIG. 6D). Illustrations show the hybridization signals in mouse brain tissue Microarray samples (FIG. 6A, FIG. 6B) and human tissue Microarray samples (FIG. 6C, FIG. 6D). Very strong hybridization signals are present in the mouse brain cortex and hypothalamus (arrows in FIG. 6 A and FIG. 6JB). The small insert in FIG. 6B is a magnification of the picture indicated by the arrow in FIG. 6B. Clear hybridization signals are present in the cells, hi the human brain, strong hybridization signals are found in the hippocampus and cortex (arrows in FIG. 6C and FIG. 6D).

FIG. 7A, FIG. 7B, FIG. 1C, FIG. 7D, FIG. 7E₅ FIG. 7F, FIG. 7G, FIG. 7H. hnmunocytochemistry analyses. Antibodies staining for cytokeratin, bcl-2, Ras, and CHAT in the tissue Microarray samples of human and mouse tissue. Cytokeratin proteins (a membrane protein) are located on the surface layer of the cells. Arrows in each panel indicate the positive signals. CHAT cells are distributed in the mucous cells of the intestine (arrow in FIG. 7A). No positive cells are found in the slides incubated without first antibody (arrow in FIG. 7E). Cytokeratin was identified in the mucous cells in the colon of cancer tissue (solid arrow in FIG. 7D). Open arrow in FIG. 7B shows lack of cytokeratin expression in the adjacent cancer cell region. Counter-staining of the adjacent section clearly shows the anatomical structure of colon cancer tissue. Solid and open arrow in FIG. 7F indicate labeled cytokeratin in mucous and cancer cell populations without labeling, respectively. Bcl-2 (arrow in FIG. 7C) and Ras (arrow in FIG. 7D) containing cells are sparsely detected in the lung cancer tissues. Staining intensities for these two proteins were low. Counter staining of immuno-stained sections showed that the cells containing Bcl-2 (arrow in FIG. 7G) and Ras (arrow in FIG. 7H) have small and round nuclei that are obviously different from the large nuclei of lung squamous cancer cells(FIG. 7H).

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

To date, human TMAs are most commonly constructed from paraffin embedded tissue blocks. The paraffin-based TMA technology is not optimal for studying RNA and proteins simultaneously on a signal array because of the degradation of RNA samples. Although a recently published OCT-embedded method improved the array in terms of RNA quality, the overall TMA configuration is not optimal and the manufacturing process is difficult. TMAs and other sections produced as described herein are made by fundamentally changing the methods used with prior paraffin-based or OCT-based tissue samples.

The presently disclosed embodiments are therefore to be considered in all respects as illustrative and not restrictive. The scope of the invention is indicated by the appended claims, rather than the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. AU patent, patent application, and literature references cited in the present specification are hereby incorporated by reference in their entirety.

EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE 1

Devices and Methods for Forming Array Recipient Blocks

As shown in FIG. 1, an exemplary system of instruments used to make a TMA recipient mold generally comprises two parts: an array cavity forming device 10, often in the form of a steel stamp with multiple steel pins, and a mold 50, often in the form of a plastic box. The system may also comprise a gel-retaining template. The diameter of the arrayed cavity in a formed recipient block will be the same as that of the pins and the number of arrayed cavities in the recipient block will be the same as that of the pins. In FIG. 1, array forming device 10 is comprised of handle 15, support or main body 20, array forming pins 30, and guide pins 40. Main body 20 may be fabricated from stainless steel, aluminum, or any other suitable material per engineering drawing specifications. In some embodiments, main body 20 is approximately 53mm x 43mm x 10mm thick and contains a boss of approximately 10mm tall x 19mm diameter and a female thread centered on the top portion of the boss for accommodating the hari_dle. The main body 20 may contain holes located as appropriate to accommodate guide pins 40. It may also contain holes located to accommodate array forming pins 30. Main ^"body 20, guides pins 40, and array forming pins 30 may be passivated prior to assembly to prevent oxidation of the instrument upon repeated cleaning and/or autoclave cycles. Handle 15 may be made of plastic or any other suitable material, hi some embodiments, tαandle 15 is about 90mm long x 26mm diameter (max), is tapered and fluted for proper ergonomic function, and contains a threaded stud insert approximately 13mm in length located at the narrow end of the handle. This allows handle 15 to be threaded onto female threads of main body 20. Guide pins 40 may be 5mm in diameter and approximately 32mm long. They can, for example be standard hardened stainless steel dowel pins and press fit into the corresponding holes of the body. Array forming pins 30 can be approximately 1.0- 1.5mm in diameter and approximately 22mm long, or of any other suitable dimensions. They may be standard hardened stainless steel dowel pins and press fit into the corresponding holes of the body.

The mold box 50 is constructed as one piece and is fabricated from polycarbonate and machined per engineering drawing specifications. It is vapor polished to> promote see-through clarity. It is approximately 53 x 43 x 26mm thick and contains a deep pocket 60 located in the geometric center of the top of the box that is approximately 37 x 28 x 13mm deep. The mold box has a wall thickness of approximately 8mm. It also contains vertical through holes 70 approximately 5mm diameter located along the outer edge of the box protruding through the wall thickness. These through holes accommodate guide pins 40 stamp and the locating pins of the gel-retaining template. The above describes a basic mold box for fabricating a single mold. For production purposes, the mold, box can be designed to accommodate multiple molds by duplicating the same configuration as described above in a matrix type fashion. The production mold box can be machined out of one piece of polycarbonate. The number of mold boxes is based on production requirements and efficiencies. A gel-retaining template is constructed as one piece and is fabricated from polycarbonate and machined per engineering drawing specifications. It is vapor polished to promote see-through clarity. It is approximately 104 x 95 x 3mm thick and contains a rectangular through hole located in the geometric center of the template that is approximately 28 x 19mm. It contains 4 integrally machined locating pins that are approximately 6.5 x 5mm diameter. These pins are located symmetrically about the main center through hole and spaced approximately 45 x 35mm apart and are designed to fit precisely into the corresponding locating holes of the mold box. The gel-retaining template also contains 4 through holes that are approximately 5mm diameter. These through holes are located symmetrically about the main center through hole and spaced approximately 45 x 35mm apart and are designed to accommodate the guide pins of the stamp. The above describes a basic gel-retaining template for fabricating a single mold. For production purposes, the gel-retaining template can be designed to accommodate multiple molds by duplicating the same configuration as described above in a matrix type fashion. The production gel-retaining template can be machined out of one piece of polycarbonate. The number of gel-retaining templates is based on production requirements and efficiencies.

EXAMPLE 2 Array Recipient Block Constructs TMA recipient blocks using the methods disclosed herein and the device described above were created.

A solution containing 13% sucrose, 0.04% paraformaldehyde, and several drops of saturated Bromphenol blue was made by mixing the above reagents with distilled water. Agarose powder was suspended into the above solution to make a suspension solution wherein the concentration of the agarose ranged from 0.05% to more than 15%. Agarose was dissolved into the solution by heating through the use of a microwave, although any other heat method could be used. The melted solution containing the agarose was poured into mold 50 and array cavity forming device 10 was placed in the solution. The solution was allowed to cool below 60⁰C to let the agarose solution coagulate. The device 10 was removed.

As shown in FIG. 2, multiple array cavities are left in the agarose in the same positions as pins 30 in array forming device 10. Those array cavities can be filled with the cored tissue samples. This array recipient block is very easy to section and tightly holds the tissue samples. Additionally, the block and its sections are easy to manipulate and allow for easy preparation of intact sections. For example, as shown in FIG. 3, no folding and crack occurred during sections.

EXAMPLE 3

Tissue Microarray Constructs

Test arrays (60 x 1.0 mm diameter samples or 96 x 1.0 mm diameter samples), using the methods disclosed herein were created. This example demonstrates how samples can be cored from fresh or fixed frozen tissue samples and placed into a recipient array block for sectioning and subsequent storage. 1.5 mm diameter Microarray steel needles can core fresh or fixed frozen tissue easily. Frozen tumor tissue and normal brain tissue were successfully cored and placed into the agarose-based array block. The array was constructed with less than lmm space between each sample.

Fresh human lung tumor tissue, normal human brain, mouse brain, and several other tissue samples were dipped into a fixative solution comprising 4% paraformaldehyde, 25-30% sucrose, and IXPBS for about two days at 0-4°C. This step is done to protect the cell morphology and prevent RNase-induced RNA degradation. The duration of this dipping varied according to the size of the tissue samples. After dipping, the fixed tissues were frozen on dry ice for 3-5 min to let the tissue become hard for being cored. Tissue samples were then punched with the tissue biopsy needles (diameter: 1.0- 1.5mm, height: 1.5-2.0cm) and placed directly into the array recipient block sitting at room temperature or in a chamber at -10°C to -15⁰C. Alternatively, frozen tissue samples can also be first embedded in gelatin on dry ice. Embedded tissue samples can be cored directly without fixation and cored tissue samples can be placed into array recipient block at room temperature or in a -10°C to -15°C chamber.

The recipient agarose block had the same size base as the paraffin recipient block that the TMA was originally made to accommodate, and therefore was easily mounted into the tissue microarrayer. Punching and coring were done slowly and with minimal pressure to prevent damage of the tissue samples. Multiple arrays were created to demonstrate the feasibility of this method. One array contained 96-cored samples including human brain, lung cancer, liver, and several other tissues. After tissue arrays were completed, the array block containing the arrayed tissue could be sectioned or wrapped and stored at —70 to -80°C freezer months or years. Sections of 4-15 microns were cut in a cryostat microtome at -20⁰C. As shown in

FIG. 4, which includes panels showing microarray method and H&E staining, an array comprising a total of 96 1.0 mm samples from human brain and other organs spaced 1.0 mm apart could be placed in agarose gel based matrix. 14-micron section of the block shown in FIG. 4 stained with H&E staining to show the overall integrity and spacing.

The fixed or unfixed tissue array samples maintained morphology when 5-15 microns sections were cut and H&E stained. FIG. 5 shows detailed morphology of tissue array, tissue cores, and cells at different magnification. The tissue array made as above invention gives rise to a defined configuration, well protected cell morphology; intact cell shape without multiple small empty holes in the sections. Similar results were obtained using a tape transfer system to section a human lung cancer array. The morphology and integrity of the tissue was comparable to that which is shown in FIG. 5. The data in this example that is the invention allows for the sectioning of other tissues that are generally difficult to section using standard methods. In a typical test array, 40-150 samples were easily placed into the array block with room to add more samples if needed. This sample size is equivalent to that seen in commercially available paraffin arrays. For example, current paraffin arrays containing up to 96 individual tissue samples and up to 200 individual samples can be purchased from different companies. To fit more samples on the frozen array, a larger agarose mold or a smaller coring needle can be used to fit more samples in the same space.

EXAMPLE 4 Non-Isotopic RNA in situ Hybridization Method Employing TMA

To demonstrate that tumor tissue and/or normal tissue containing TMAs can be used for analysis of RNA molecules and proteins, non-radioactive RNA in situ hybridization was performed on a TMA slide using a fluorescein-labeled mouse vasoactive intestinal peptide (VIP) RNA and human GAPDH RNA antisense probe.

Array slides were washed in 6M NaI for 1-5 min to remove agarose. After washing, tissue arrays underwent standard in situ hybridization experimental protocols. Non-isotopic RNA in situ hybridization was performed as previously published for frozen sections (Waschek et al, 1998). Slides were washed in PBS (Ambion Inc., Cat# 9624) for 3 x 5 min. Sections were covered with pre-hybridization buffer and placed in a humid chamber at 50-55⁰C for 2 to 4 hours. Hybridization was done by adding 300-500ng/ml fluorescein-labeled VIP or GAPDH RNA antisense probes. The antisense probes are made based on the protocol published elsewhere (Waschek et al, 1998). A sense probe was used as the negative control. Hybridization was done at 50- 55⁰C overnight. Slides were then washed with 4XSSC and 2XSSC, each for 10 min at 50-55°C. RNase A/Tl treatment was performed on the slides at room temperature to remove the nonspecific probe. After washing in 0.2XSSC at 50-55⁰C, the slides were dipped in PBST (0.1% triton in IXPBS) for 30 min. Slides were then incubated with the anti-fluorescein-AP-conjugates for 4 hours at room temperature. Excess antibodies were then washed with IXPBST 4 times each for 20 min. The last wash could be done overnight if necessary. Color staining was performed by dipping the slides into a solution containing a NBT/BCIP mixture. Slides were post-fixed in 4% paraformaldehyde in PBS and the signal was visualized using standard light microscopy. As shown in FIG. 6, strong hybridization signals for VTP and GAPDH were present in the array samples.

EXAMPLE 5

Immunohistochemical Methods Employing Tissue Microarray

Immunocytochemical methods can be easily performed on TMAs prepared according to the invention, as in the study described below where immunohistochemistry was performed on a tumor TMA using an antibody against cytokeratin, Ras, Bcl-2 and CHAT.

Array slides for immunocytochemistry were prepared by sectioning off the block as described above. Sections were rinsed in Ix PBS₅ and quenched in 2.0% hydrogen peroxide for 30 min to kill the endogenous peroxidases. Imrnunocytochemistry was performed using standard procedures. Slides were incubated with 2% normal goat serum for 1-2 hours. Monoclonal antibody anti-cytokeratin (Chemicon, Cat#MAB3412) at the dilution of 1:10,000 was incubated with the sections overnight at room temperature. The primary antibodies were not included in the negative control experiments. Slides were incubated with the secondary antibodies (biotinylated anti-mouse IgG: Vector Lab. Cat# PK-6102). Solution A and B (Vector Inc, ABC kit, Cat# PK-6102) were mixed and then added on slide for 30 min. DAB (Vector Lab, Cat# SK-4100) was used as a chromogen, and arrays were visualized and photographed using standard light microscopy.

As can be seen in FIG. 7, each of the representative protein markers was successfully detected in the human tumor TMA slides. These results demonstrate that the present invention will create an array with a high quality of preserved proteins. H: * * * H= *

AU of the methods, tissue sample recipient blocks, fixative solutions, solutions, sections, kits, and systems disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the methods, tissue sample recipient blocks, fixative solutions, solutions, sections, kits, and systems of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods, tissue sample recipient blocks, fixative solutions, solutions, sections, kits, and systems described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

U.S. Patent 6,696,271 B2

Andersen et al., Am. J. Pathol, 161:73-79, 2002.

Battifora, Lab. Invest., 55:24-28, 1986.

Battifora et al, Lab. Invest., 63:722-724, 1990.

Bubendorf et al, Cancer Res., 59:803-806, 1999a.

Bubendorf et al., J. Natl. Cancer Institute, 91:1758-1764, 1999b.

Dan et al, World J. Gastro., 10:579-582, 2004.

Fejzo et al, American J. Pathol, 5:1645-1650, 2001.

Goldsworthy et al, Molecular Carcinogenesis, 15:86-91, 1999.

Hsu et al, Mod. Pathology, 15:1374-1380, 2002.

Kononen et al Nature Medicine, 4:844-847, 1998.

Mousses et al, Curr. Opin. Chem. Biol, 6:97-101, 2002.

Moch et al, Verhandlungen der Deutschen Gesellschaft fur Pathologie, 83:225-232,

1999.

Schrami et al, Clinical Cancer Research, 5(8):1966-1975, 1999. Shergill et al, Curr. Med. Res. Opin., 5:707-712, 2004. Simon et al, J. Natl. Cencer lnst., 93(15):1141-1146, 2001. Sambrook et al, In: Molecular cloning, Cold Spring Harbor Laboratory Press, Cold Spring

Harbor, NY, 1989.

Varambally et al, Nature, 419:624-629, 2002. Waschek et al, Proc. Natl Acad. ScL USA, 95:9602-9607, 1998.

Claims

CLAIMS:

1. A tissue sample recipient block comprising a matrix material and a cryostat protection component, said block having at least one array cavity disposed therein.

2. The tissue sample recipient block of claim 1, further defined as a microarray recipient block.

3. The tissue sample recipient block of either of claims 1 or 2, further defined as comprising a plurality of array cavities.

4. The tissue sample recipient block of any of claims 1 to 3, further defined as comprising at least 10 array cavities.

5. The tissue sample recipient block of any of claims 1 to 4, further defined as comprising at least 96 array cavities.

6. The tissue sample recipient block of any of claims 1 to 5, wherein the matrix material is any type of agarose.

7. The tissue sample recipient block of any of claims 1 to 6, further defined as comprising 0.1% to 15% agarose.

8. The tissue sample recipient block of any of claims 1 to 7, further defined as comprising 0.3% to 12% agarose.

9. The tissue sample recipient block of any of claims 1 to 8, further defined as comprising 0.5% to 10% agarose.

10. The tissue sample recipient block of any of claims 1 to 9, wherein the cryostat protection component is a saccharide, glycerol, alcohol, or OCT.

11. The tissue sample recipient block of any of claims 1 to 10, wherein the cryostat protection component is a saccharide.

12. The tissue sample recipient block of any of claims 1 to 11 , wherein the saccharide is sucrose.

13. The tissue sample recipient block of any of claims 1 to 12, further defined as comprising between 5% and 25% sucrose.

14. The tissue sample recipient block of any of claims 1 to 13, further defined as comprising between 10% and 20% sucrose.

15. The tissue sample recipient block of any of claims 1 to 14, further defined as comprising between 10% and 15% sucrose.

16. The tissue sample recipient block of any of claims 1 to 15, further defined as comprising about 13.5% sucrose.

17. The tissue sample recipient block of any of claims 1 to 16, further defined as comprising an antimicrobial agent.

18. The tissue sample recipient block of any of claims 1 to 17, wherein the antimicrobial agent is paraformaldehyde.

19. The tissue sample recipient block of any of claims 1 to 18, further defined as comprising 0.01% to 0.1% paraformaldehyde.

20. The tissue sample recipient block of any of claims 1 to 19, further defined as comprising 0.01% to 0.04°/ό paraformaldehyde.

21. The tissue sample recipient block of any of claims 1 to 20, further defined as comprised in a package.

22. The tissue sample recipient block of any of claims 1 to 21, wherein the package is further defined as a sealed package that prevents drying of the tissue sample recipient block.

23. The tissue sample recipient block of any of claims 1 to 22, wherein the package is further defined as a plastic box.

24. The tissue sample recipient block of any of claims 1 to 23, further defined as comprised in a kit.

25. A fixative solution comprising a fixative and a cryostat protection component.

26. The solution of claim 25, wherein the fixative is paraformaldehyde, formaldehyde, buffer formalin, glutaradehyde, ethanol, or acetone.

27. The solution of either of claims 25 or 26, wherein the fixative is paraformaldehyde.

28. The solution of any of claims 25 to 27, further defined as comprising 1% to 10% paraformaldehyde.

29. The solution of any of claims 25 to 28, further defined as comprising 3% to 6% paraformaldehyde.

30. The solution of any of claims 25 to 29, further defined as comprising about 4% paraformaldehyde.

31. The solution of any of claims 25 to 30, wherein the cryostat protection component is a saccharide, glycol, alcohol, or OCT.

32. The solution of any of claims 25 to 31 , wherein the cryostat protection component is a saccharide.

33. The solution of any of claims 25 to 32, wherein the saccharide is sucrose.

34. The solution any of claims 25 to 33, further defined as comprising phosphate buffered saline.

35. The solution of any of claims 25 to 34, wherein the cryostat protection component is a saccharide, glycerol, alcohol, or OCT.

36. The solution of any of claims 25 to 35, wherein the cryostat protection component is a saccharide.

37. The solution of any of claims 25 to 36, wherein the saccharide is sucrose.

38. The solution of any of claims 25 to 37, further defined as comprising between 10 and 50% sucrose.

39. The solution of any of claims 25 to 38, further defined as comprising between 15% and 40% sucrose.

40. The solution of any of claims 25 to 39, further defined as comprising between 20% and 35% sucrose.

41. The solution of any of claims 25 to 40, further defined as comprising about 30% sucrose.

42. The solution of any of claims 25 to 41 , further defined as a solution that preserves cellular morphology in a frozen or fresh sample fixed in the solution.

43. The solution of any of claims 25 to 42, further defined as a solution that preserves RNA quality and/or quantity in a sample fixed in the solution.

44. The solution of any of claims 25 to 43, further defined as comprised in a kit.

45. A method of preparing a tissue sample comprising block, the method comprising: obtaining a tissue sample recipient block of any of claims 1 to 24; obtaining at least one tissue sample that is unfixed or has been fixed in a solution of any of claims 25 to 44; and placing the sample in the block.

46. The method of claim 45, further defined as comprising obtaining a plurality of samples that are unfixed or have been fixed in the solution and placing the plurality of samples individually in a plurality of array cavities in the block.

47. The method of either of claims 45 or 46, further defined as comprising placing at least 10 samples in at least 10 array cavities in the block.

48. The method of any of claims 45 to 47, further comprising sectioning the block to obtain a section.

49. The method of any of claims 45 to 48, further defined as a method of preparing a tissue microarray.

50. The method of any of claims 45 to 49, wherein sectioning comprises sectioning a 1 μm to 20 μm thick section comprising a portion of the sample.

51. The method of any of claims 45 to 50, wherein the section is 2 μm. to 10 μm thick.

52. The method of any of claims 45 to 51, wherein the section is about 4 μm thick.

53. The method of any of claims 45 to 52, wherein the cryotstat protection component in the block facilitates sectioning of the block to produce an intact section comprising a portion of the sample surrounded by matrix.

54. The method of any of claims 45 to 53, further comprising performing an in situ hybridization on the section.

55. The method of any of claims 45 to 54, further comprising performing immunohistochemistry on the section.

56. The method of any of claims 45 to 55, further comprising dehydrating the section.

57. The method of any of claims 45 to 56, further comprising analyzing RNA in the section.

58. The method of claim 57, further comprising isolating RNA from the section.

59. The method of any of claims 45 to 56, further comprising analyzing DNA in the section.

60. The method of claim 59, further comprising isolating DNA from the section.

61. The method of any of claims 45 to 56, further comprising analyzing protein in the section.

62. The method of claim 61 , further comprising isolating protein from the section.

63. The method of any of claims 45 to 62, wherein the first tissue sample is frozen.

64. The method of any of claims 45 to 63, wherein the sample has been frozen and then fixed.

65. The method of any of claims 45 to 64, wherein the sample is further defined as an archival sample.

66. The method of any of claims 45 to 63, wherein the sample has been fixed and then frozen.

67. The method of any of claims 45 to 66, further comprising obtaining the sample from a live or dead organism.

68. The method of any of claims 45 to 67, wherein obtaining the first tissue sample comprises obtaining a core of tissue that fits within one of the array cavities.

69. The method of any of claims 45 to 68, further comprising making the block by placing liquid matrix in container, placing a device comprising a plurality of pins in the liquid matrix, and allowing the matrix to solidify.

70. A section prepared by a method set forth in claims 45 to 69.

71. The section of claim 70, further defined as a tissue microarray.

72. The section of either of claim 70 or 71 , further defined as comprising a plurality of tissue samples disposed in said matrix.

73. The section of any of claims 70 to 72, further defined as comprising at least 10 tissue samples disposed in said matrix.

74. The section of any of claims 70 to 73, wherein the matrix comprises at least 96 tissue samples disposed in said matrix.

75. The section of any of claims 70 to 74, further defined as 1 μm to 20 μm thick.

76. The section of any of claims 70 to 75, further defined as 2 μm to 10 μm thick.

77. The section of any of claims 70 to 76, further defined as about 4 μm thick.

78. The section of any of claims 70 to 77, further defined as comprised in a kit.

79. A kit for practicing the method of claims 45 to 69, comprising: a microarray recipient block of any of claims 1 to 24; and a fixative solution of any of claims 25 to 44.

80. The kit of claim 80, further defined as comprising a punch for preparing a core tissue sample.

81. A kit for practicing the method of any of claims 45 to 69, comprising: a matrix material; a cryostat protection component; and a fixative solution of any of claims 25 to 44.

82. The kit of claim 82, further defined as comprising one or more components of a system for making a microarray recipient block comprising the matrix material and cryostat protection component.

83. A system for forming a tissue sample recipient block of any claims 1 to 24, said block having at least one array cavity disposed therein, the system comprising at least an array cavity forming device comprising at least one member configured to form an array cavity in the matrix material operably disposed on a support.

84. The system of claim 83, further defined as a system for preparing a microarray recipient block.

85. The system of either of claims 83 or 84, wherein the at least one member is further defined as a steel pin.

86. The system of any of claims 83 to 85, wherein the steel pin is of a diameter of from 0.6 to 1.0 mm.

87. The system of any of claims 83 to 86, wherein the steel pin is 1.0 to 2.5 cm long.

88. The system of any of claims 83 to 87, wherein the steel pin is 1.5 to 2.0 cm long.

89. The system of any of claims 83 to 83, further defined as comprising a plurality of members operably arrayed on the support.

90. The system of any of claims 83 to 89, further defined as comprising at least 10 members.

91. The system of any of claims 83 to 90, further defined as comprising at least 96 members.

92. The system of any of claims 83 to 89, wherein the plurality of members are arrayed on a 1.0 to 4.0 cm by 1.0 to 4.0 cm region of the support.

93. The system of any of claims 83 to 92, wherein the plurality of members are arrayed on a 1.5 to 2.0 cm by 1.5 to 2.0 cm region of the support.

94. The system of any of claims 83 to 93, wherein the plurality of members are arrayed on an about 1.5 cm by about 2.0 cm region of the support.

95. The system of any of claims 83 to 94, further defined as comprising a handle operably coupled to the support.

96. The system of any of claims 83 to 95, wherein the handle is coupled to the support on a side opposite the at least one member.

97. The system of any of claims 83 to 96, further defined as comprising a mold into which liquid matrix material and the array cavity forming device can be introduced during use.

98. The system of any of claims 83 to 97 wherein the mold is plastic.

99. The system of any of claims 83 to 98, wherein the plastic is polycarbonate.

100. The system of any of claims 83 to 99, wherein the mold is a box of 1.0 to 5.0 cm by 1.0 to 5.0 cm.

101. The system of any of claims 83 to 100, wherein the mold is a box of about 3.0 cm by about 4.0 cm.

102. A method of preparing a tissue sample comprising block, the method comprising: obtaining a tissue sample recipient block comprising a matrix material and a first cryostat protection component, said block having at least one array cavity disposed therein; obtaining at least one tissue sample that is unfixed or has been fixed in a solution comprising a fixative and a second cryostat protection component; and placing the sample in an array cavity in the block.

103. The method of claim 102, further defined as comprising obtaining a plurality of samples that are unfixed or have been fixed in the solution and placing the plurality of samples individually in a plurality of array cavities in the block.

104. The method of claim 102, wherein the block comprises a plurality of array cavities.

105. The method of claim 102, wherein the matrix material comprises agarose.

106. The method of claim 105, wherein the block comprises 0.1% to 15% agarose.

107. The method of claim 102, wherein the first cryostat protection component and/or second cryostat protection component is a saccharide, glycerol, alcohol, or OCT.

108. The method of claim 107, wherein the first cryostat protection component and/or second cryostat protection component is sucrose.

109. The method of claim 108, wherein the block comprises between 5% and 25% sucrose.

110. The method of claim 108, wherein the solution comprises between 10% and 50% sucrose.

111. The method of claim 102, wherein the block comprises an antimicrobial agent.

112. The method of claim 111, wherein the antimicrobial agent is paraformaldehyde.

113. The method of claim 102, wherein the fixative is paraformaldehyde, formaldehyde, buffer formalin, glutaradehyde, ethanol, or acetone.

114. The method of claim 113, wherein the fixative is paraformaldehyde.

115. The method of claim 114, wherein the second cryostat protection component is sucrose.

116. The method of claim 102, wherein the matrix is agarose, the first cryostat protection component is sucrose, the fixative is paraformaldehyde, and the second cryostat protection component is sucrose.

117. The method of claim 102, further comprising sectioning the block to obtain a section.

118. The method of claim 117, further defined as a method of preparing a tissue microarray.

119. The method of claim 102, wherein the first tissue sample is frozen.

120. The method of claim 102, wherein obtaining the first tissue sample comprises obtaining a core of tissue that fits within one of the array cavities.

121. The method of claim 102, further comprising making the block by placing liquid matrix in container, placing a device comprising a plurality of pins in the liquid matrix, and allowing the matrix to solidify.

122. A tissue sample recipient block comprising a matrix material and a cryostat protection component, said block having at least one array cavity disposed therein.

123. The tissue sample recipient block of claim 122, further defined as a microarray recipient block.

124. The tissue sample recipient block of claim 122, further defined as comprising a plurality of array cavities.

125. The tissue sample recipient block of claim 122, wherein the matrix material comprises agarose.

126. The tissue sample recipient block of claim 125, wherein the cryostat protection component is a saccharide, glycerol, alcohol, OCT.

127. The tissue sample recipient block of claim 126, wherein the cryostat protection component is sucrose.

128. The tissue sample recipient block of claim 122, further defined as comprising an antimicrobial agent.

129. The tissue sample recipient block of claim 128, wherein the antimicrobial agent is paraformaldehyde.

130. A fixative solution comprising a fixative and a cryostat protection component.

131. The solution of claim 130, wherein the fixative is paraformaldehyde, formaldehyde, buffer formalin, glutaradehyde, ethanol, or acetone.

132. The solution of claim 130, wherein the fixative is paraformaldehyde.

133. The solution of claim 131, wherein the cryostat protection component is a saccharide, glycol, alcohol, or OCT.

134. The solution of claim 133, wherein the cryostat protection component is sucrose.

135. A section comprising:

(a) a matrix comprising a matrix material and a first cryostat protection component; and

(b) at least one tissue sample that has been fixed in a solution comprising a fixative and a second cryostat protection component disposed within said matrix.

136. The section of claim 135, further defined as a tissue microarray.

137. The section of claim 135, wherein the matrix material comprises agarose.

138. The section of claim 135, wherein the first cryostat protection component and/or second cryostat protection component comprises a saccharide, glycerol, alcohol, or OCT.

139. The section of claim 138, wherein the first cryostat protection component and/or second cryostat protection component is sucrose.

140. The section of claim 135, wherein the matrix comprises an antimicrobial agent.

141. The section of claim 135, wherein the fixative is paraformaldehyde, formaldehyde, buffer formalin, glutaradehyde, ethanol, or acetone.

142. A kit comprising: a microarray recipient block comprising a matrix material and a first cryostat protection component, said block having a plurality of array cavities disposed therein; and a fixative solution comprising a fixative and a second cryostat protection component.

143. A system for forming a tissue sample recipient block comprising a matrix material, said block having at least one array cavity disposed therein, the system comprising at least an array cavity forming device comprising at least one member configured to form an array cavity in the matrix material operably disposed on a support.