RAPID COMMUNICATION |
Resin Tissue Microarrays : a Universal Format for Immunohistochemistry
Atlas of Protein Expression Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, United Kingdom
Correspondence to: Dr. W.J. Howat, Atlas of Protein Expression Project, Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1HH, UK. E-mail: wjh{at}sanger.ac.uk
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Summary |
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Key Words: tissue microarray resin TMA glycol methacrylate immunohistochemistry single-chain Fv
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Introduction |
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Resin embedding has been used as a technique to improve resolution of fine structural detail for many years, particularly in electron microscopy. Two types of resins have been utilized, either epoxy or acrylic based. Epoxy resins, such as Araldite, Epon, and Spurr (Glauert et al. 1956; Kushida 1959
; Spurr 1969
), are highly hydrophobic and therefore require complete dehydration of the tissue before embedding, and polymerize above 50C. Tissue fixed and processed through this medium is often unsuitable for immunohistochemistry (IHC). Acrylate resins have emerged as the best medium for tissue and antigen preservation for light or electron microscopy, inasmuch as they are water miscible and can be polymerized at lower temperatures with the addition of a suitable catalyst. Of the many acrylate resins available, 2-hydroxyethyl methacrylate (HEMA, glycol methacrylate or GMA) have been used extensively for IHC, including the study of rat fetal bone (Onetti Muda et al. 1992
) and human colon (Mozdzen and Keren 1982
), in biopsies of conjunctiva (Ahluwalia et al. 2001
) and bronchus (Wilson et al. 2001
), as well as in routine bone marrow pathology (Beckstead et al. 1981
; Westen et al. 1981
). GMA embedding involves the infiltration of GMA monomer into and between the tissue elements, followed by embedding in GMA monomer, plasticizer, an initiator, and an accelerator; the initiator and accelerator act to drive the free radicalmediated polymerization of the GMA monomer (Gerrits and Horobin 1996
). Addition of a plasticizer prevents a brittle GMA block and aids in cutting. The advantages that resin embedding has over conventional frozen and paraffin embedding media include thinner section cutting, resulting in an increase in optical clarity of the section, reproducible section thickness, and fixative-dependent antigen preservation (Casey et al. 1988
; Hand et al. 1996
).
Despite the existence of resin-embedding methods for many years and the application of TMA for efficient staining of multiple samples simultaneously, this study represents the first demonstration of resin embedding for TMA. This embedding method has the potential to provide superior morphological detail while retaining antigenic capacity.
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Materials and Methods |
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Acetone Fixation
Acetone containing protease inhibitors, 20 nM iodoacetamide and 2 mM phenylmethyl sulfonyl fluoride (Sigma; Poole, UK) was prepared at least 24 hr before use and stored at 20C. Tissues were immersed in this fixative at 20C immediately after excision and fixed for 16 hr to 1 week, depending on the experiment.
Formalin Fixation
Tissues were immersed in 10% neutral buffered formalin (BDH; Poole, UK) and fixed at room temperature for 48 hr before three changes in 70% ethanol (2 hr each), and two changes in 100% ethanol, with the final change left overnight.
Array Manufacture
The Beecher Instruments Manual Tissue Arrayer (MTA1; Beecher Instruments, Inc., Sun Prairie, WI) was used in the construction of the arrays. Working under a fume hood, we made a 2% agarose/H2O gel using electrophoresis-grade agarose (Invitrogen; Paisley, UK) and set it in a paraffin wax embedding mold. The resultant agarose block was trimmed to 5-mm to 6-mm thickness to fit into the MTA1 recipient block holder. This was immersed in acetone to prevent drying of the subsequent array. A 0.6-mm donor needle was used to disperse the agarose and create a hole at the desired start position.
Tissues fixed in 20C acetone were warmed to room temperature for 15 min. Once warmed, a tissue was cored with the 0.6-mm donor needle while immersed in acetone then placed into the recipient hole, and a fresh recipient hole was made. A 0.2-mm gap was used between each core. For formalin-fixed tissue, the same methodology was applied but with immersion in ethanol rather than acetone and with the warming step excluded.
Following completion of the array, excess agarose was removed and the agarose array block immersed in methyl benzoate for 1 hr at room temperature with rotation (Britten et al. 1993). Subsequent infiltration of the agarose array block was performed overnight at 4C in GMA monomer (JB4 kit; Park Scientific, Northampton, UK) containing 5% methyl benzoate, with a minimum of three changes of infiltration solution. The agarose array block was embedded in Taab flat-bottomed electron microscopy molds (Taab Laboratories; Aldermaston, UK) in catalyzed GMA monomer (10 ml GMA monomer with 0.07 g benzoyl peroxide), with N,N-dimethylaniline added as an accelerator. These were left to polymerize for a minimum of 2 days at 4C. Long-term block storage was at 20C to prevent further polymerization.
Two-µm array sections were cut at room temperature using a glass knife on a Leica RM2165 semi-thin microtome (Leica; Milton Keynes, UK). The sections were floated onto a room temperature water bath containing 1% ammonia solution, picked up on SuperFrost Plus glass slides (BDH), and air dried for a minimum of 1 hr before long-term storage at 80C (although any temperature below freezing should be sufficient to prevent further polymerization of the cut section). To examine general tissue morphology, sections were stained with Mayers hematoxylin.
Frozen Tissue Microarrays
Frozen TMA sections were purchased from Covance (Covance Ltd.; Harrogate, UK), with blocks constructed by Covance from the same murine strain as used in-house.
Paraffin Tissue Microarrays
Paraffin TMA blocks were made in-house on the automated tissue arrayer (ATA-27; Beecher Instruments) from tissue, formalin-fixed for 48 hr, and processed into Paraplast (Thermo Shandon; Runcorn, UK). Four-µm sections were cut on a Leica RM2125RT rotary microtome and floated onto water at 4045C. Sections were picked up on SuperFrost Plus slides and dried at 4045C overnight.
Immunohistochemistry
Automated IHC was performed on the Ventana Discovery platform (Ventana Medical Systems; Tucson, AZ) using proprietary reagents at 37C, unless otherwise specified. Resin array sections were warmed to room temperature for 30 min before staining. Antigen retrieval, required for formalin-fixed arrays, used mild Cell Conditioner 1 (Tris/EDTA/borate buffer, pH 8), or a combination of Cell Conditioner 1 plus protease retrieval with an alkaline protease (0.02 units/ml for 10 min). Antigen retrieval was not required for acetone-fixed preparations.
Primary antibodies used were monoclonal rat anti-mouse CD45/B220 (Clone RA3-6B2; R and D Systems, Abingdon, UK), monoclonal rabbit anti-mouse Ki-67 (Clone SP6; Lab Vision Corporation, Fremont, CA), and polyclonal rabbit anti-laminin (Sigma). Sections with no addition of primary antibody were used as negative controls. Biotinylated species-specific secondary antibodies, cross-absorbed against murine immunoglobulin, were obtained from Jackson Laboratories (Jackson Immunoresearch Labs; West Grove, PA).
For resin TMAs, primary antibodies were incubated for 8 hr and secondary antibodies for 1 hr. Detection of antibody binding was with a peroxidase-labeled streptavidin-biotin technique with diaminobenzidine plus copper enhancement (DABMap kit, Ventana). Hematoxylin was used as a nuclear counterstain. Inhibition of endogenous peroxidase and blocking of nonspecific binding sites were included as part of the DABMap detection.
Paraffin IHC was undertaken using the same methodology as that used for formalin-fixed resin IHC, with the addition of deparaffinization on the Ventana Discovery using the proprietary solution (EZPrep). Primary antibodies were incubated for 20 min and secondary antibodies for 8 min.
Frozen IHC was undertaken using the same methodology as that used for acetone-fixed resin IHC, with the addition of acetone fixation for 15 min at room temperature. Primary antibodies were incubated for 20 min and secondary antibodies for 8 min.
Staining was also performed with in-housegenerated single-chain Fv (scFv) preparations against human desmin coupled to a Tri FLAG tag. ScFvs were applied for 4 hr and detected using a biotinylated anti-FLAG for 30 min (Sigma) followed by tyramine signal enhancement with the Ventana Tyramide Signal Amplification kit (AmpMap). This utilized a streptavidin-peroxidase conjugate, dinitrophenol-labeled tyramine, and biotinylated detection of dinitrophenol. All detection steps were controlled with the AmpMap kit and Ventana software. Color development used diaminobenzidine. Sections with no addition of primary antibody were used as negative controls.
Image Capture
All images were captured using a Zeiss Axioskop2 using a 40x NeoFluor lens through an HRc color camera (Zeiss; Welwyn Garden City, UK) and coupled to Axiovision 4 (Imaging Associates; Bicester, UK).
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Results |
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Using this agarose block and coring method, a 20-tissue TMA was constructed. First, a hole was made in the recipient agarose block. Second, a "composite core" was constructed by repeated coring of the fixed tissue with resultant build-up of the composite core in the donor needle. This was then extruded into the recipient hole. Area selection was performed at the gross tissue level and all tubular tissues, e.g., intestine, esophagus, and trachea, were cored in the transverse direction. The length of the composite core could be regulated by the number of repeated cores taken from a tissue but was limited to the size of the tissue available.
The 20-tissue TMA (0.6-mm core with 0.2-mm gap between cores) measured 3.8 mm x 3 mm, with additional surrounding agarose. The 0.6-mm cores shrank during the infiltration and embedding process, resulting in a diameter of 400500 µm. This was considerably smaller than a similar commercially available 20-tissue frozen TMA (1.5-mm core with 0.5-mm gap) at 9.5 mm x 7.5 mm. Imaging by stereo microscopy showed the precision of the arraying process with agarose and the permeation of the agarose transfer medium with resin (Figure 1A). Where cores bent toward the end of the array, this could also be seen.
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IHC of acetone-fixed resin TMAs with the extracellular matrix antigen laminin and mouse B lymphocyte marker CD45 (B220) showed superior morphology and resolution when compared with similarly treated acetone-fixed frozen TMA sections (Figures 2A2D). CD45 on resin spleen showed a clear cell surface localization of the antibody staining, with distinct hematoxylin staining of the nuclear compartment (Figure 2A). In contrast, although an enhanced intensity could be received from frozen spleen, distinct cell surface staining was lost, with a "blush" over the cytoplasmic and nuclear compartments, and nuclear morphology with hematoxylin was compromised (Figure 2B). A similar pattern could be seen with laminin staining in cerebellum, where clear basement membrane localization of endothelial vessels on resin (Figure 2C) was sharply contrasted, with intense but undefined staining in frozen tissue (Figure 2D). Furthermore, frozen tissue demonstrated loss of neuropil in the molecular layer, which was retained in resin tissue. Ki-67, CD138, and myeloperoxidase have also been demonstrated in acetone-fixed resin TMA sections, but no staining has been demonstrated with any negative controls (data not shown).
Comparison among resin, frozen, and paraffin TMAs of kidney stained with laminin demonstrated that there was equivalent signal localization to the basement membrane with all three formats (Figures 3A3C). The fine structural detail of laminin expression in the basement membrane of the glomerular tuft and Bowman's capsule was resolved with exquisite clarity using the resin TMA (Figure 3A). In contrast, the intensity of signal, combined with the thickness of the frozen and paraffin TMA sections, could not resolve the detail of the network of vessels (Figures 3B and 3C).
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Formalin-fixed Resin TMAs
TMAs have largely been constructed using formalin-fixed material; therefore, it was important to confirm that the resin format could also be used for tissue fixed in 10% neutral buffered formalin. Formalin fixation alone did not result in tissue rigidity sufficient for direct tissue coring; therefore, the tissue was dehydrated using ethanol. The rigidity that this conferred allowed cores to be taken from the tissue; however, re-arraying into the agarose immersed in ethanol led to some dispersion of the formalin-fixed cores and consequent loss of distinction between cores (not shown).
IHC performed on the formalin-fixed resin TMA confirmed that antigens that could be visualized on FFPE TMA could also be visualized on formalin-fixed resin TMA, using the same antigen retrieval techniques (Figures 4C and 4D). Intensity of signal for laminin staining of kidney was equivalent to FFPE TMA (Figure 3C), but resolution of the staining pattern was improved. Ki-67 staining of spleen showed intense nuclear localization and clear identification of proliferating lymphocytes.
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Discussion |
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TMAs made by this resin-embedding method demonstrated an overall improvement of tissue morphology when compared with similar high-quality frozen TMAs from a commercial supplier. Liver and spleen proved difficult to process, and some loss of morphology was shown; however, following immunohistochemistry, morphology was comparable and, in most cases, better than that of frozen TMAs. Antigen preservation in acetone-fixed resin TMAs was equivalent to that in frozen TMAs: all antigens tested localized to the same cellular and extracellular compartments in both resin-embedded TMAs and frozen TMAs. Thus, acetone-fixed resin TMAs are an improvement on the current frozen TMA methodology.
The construction of the TMA was only made possible by the addition of an intermediary "medium" of a 2% agarose gel. This allowed accurate positioning of donor tissues into the recipient array block, as is performed during paraffin arraying, while also allowing resin penetration and polymerization. However, the change in density that occurred while cutting through resin-infiltrated agarose and through resin-infiltrated tissue resulted in occasional folds in the tissue. Agarose has been used as a medium for re-positioning of specimens in the processing of Zebrafish embryos for histology (Tsao-Wu et al. 1998) and in cell preparations for IHC (Kerstens et al. 2000
) and electron microscopy (Widehn and Kindblom 1990
; Mansy 2004
). A number of other methods were attempted for arraying, including the processing of individual cores and re-arraying using tape (Golick and Federman 1985
), or re-arraying into a blank resin block into which a hole had been drilled and re-polymerizing the block, a method similar to that used in frozen arraying (Miyaji et al. 2002
). Although these methods produced a workable resin array, the resulting morphology and ease of production of the current agarose method was superior.
It is a requirement of frozen arraying that 0.6-mm to 1.5-mm cores be used, and the recommended gap is 1 mm (Fejzo and Slamon 2001). In contrast, the resin format allowed a 0.6-mm core with a 0.2-mm gap to be arrayed precisely, and thus the density of cores within a resin TMA for a given size is greater than for frozen TMA. With the 7-mm physical limitation of the embedding mold used, the theoretical number of arrayed cores of 0.6 mm with a 0.2-mm gap would be 8 x 8, 64 cores. The use of glass Ralph knives (Bennett et al. 1976
) and custom-built embedding molds (Beeckman and Viane 2000
) could allow larger arrays to be built and cut. In combination with the small size of the arrays, multiple different resin array sections could be placed on a single slide, thus providing sufficient material for a validated study (Agaton et al. 2003
). Furthermore, the density of resin allows 2-µm sections to be cut, in comparison to 7- to 10-µm for frozen and 4-µm for paraffin. Thus, arraying in resin potentially provides two to five times more sections than conventional formats for the same depth of block.
TMAs have largely been constructed using formalin-fixed material; therefore, it was important to confirm that the resin format could be also be used for formalin-fixed tissue. Although the formalin fixation route provided some difficulty in the arraying process, the resultant array demonstrated outstanding preservation of fine structural and subcellular detail while providing immunostaining profiles and signal intensity comparable to those of FFPE TMA. Neither protease nor heat-mediated antigen retrieval techniques (Shi et al. 1991) affected the tissue morphology of the formalin-fixed resin TMA. Therefore, resin embedding can be considered to be an alternative TMA format for both conventional frozen and formalin-fixed TMA formats.
TMAs of conventional formats have also been used for in situ hybridization as well as immunohistochemistry (Al-Kuraya et al. 2004). Although resin TMAs have not been processed using this method, there is evidence that fixation by formalin or glutaraldehyde and embedding in acrylate resin allows the demonstration of DNA and mRNA transcripts (Le Guellec et al. 1992
; Morey et al. 1995
).
Recent advances in selection of recombinant antibodies have provided a rich potential source of antibodies for IHC. Using phage display, the genes encoding antibody fragments of desired specificity can be selected from large libraries by panning against the desired antigen. Once the gene is isolated, antibody product (usually in the form of scFvs or Fab fragments) can be generated by expression in bacterial cells. Using this E. colibased method, selection and subsequent screening on many different antigens can be carried out in parallel. The scFv format was tested on tissue arrayed on acetone-fixed resin TMA, and appropriate muscle fiber staining was observed with an anti-desmin antibody fragment. This result represents an important step in the integration of acetone- and formalin-fixed resin TMAs into high-throughput immunohistochemistry (Warford 2004).
Although the advantages that resin arraying lends to TMA are clear, there are other factors to be considered before arraying by this method. Area identification was performed at the gross anatomical level only, because sections from the tissues before coring could not be taken. For the normal tissue set used for the construction of the current resin TMA, this did not present a problem; however, this limitation may impact the reliability of coring from diseased tissues. Coring of tubular tissues such as intestine had to be conducted in the transverse directionacross the muscularis mucosa, through sub-mucosa into mucosa and intestinal lumen and vice versa. Thus, representation of the entirety of the murine intestine in one section was lost. Multiple coring of the same tissue was used throughout the array construction process to increase the depth and thus number of sections from the array. Because there is no supporting medium surrounding the tissue, tissues of low density, such as lung, or with a high percentage of loose connective tissue, such as intestine, showed some compression and consequent loss of tissue architecture. All of the above issues could be circumvented with the introduction of a supporting medium to the tissue after fixation, akin to paraffin arraying, allowing re-orientation of tubular tissues and section-aided coring. Finally, the technology is currently limited to freshly collected tissue. For the purposes of prospectively collected tissue, this does not pose a problem, but currently does exclude use from archived material.
In conclusion, resin TMAs combine the superior morphology of formalin-fixed material with the antigenicity of fresh frozen acetone-fixed material in one flexible format that conserves array material through thin sectioning. The advantages that resin TMA provides over and above other TMA formats lends the technology to other potential applications in pharmaceutical research and safety testing of antibodies.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Agaton C, Galli J, Hoiden Guthenberg I, Janzon L, Hansson M, Asplund A, Brundell E, et al. (2003) Affinity proteomics for systematic protein profiling of chromosome 21 gene products in human tissues. Mol Cell Proteomics 2:405414
Ahluwalia P, Anderson DF, Wilson SJ, McGill JI, Church MK (2001) Nedocromil sodium and levocabastine reduce the symptoms of conjunctival allergen challenge by different mechanisms. J Allergy Clin Immunol 108:449454[CrossRef][Medline]
Al-Kuraya K, Schraml P, Torhorst J, Tapia C, Zaharieva B, Novotny H, Spichtin H, et al. (2004) Prognostic relevance of gene amplifications and coamplifications in breast cancer. Cancer Res 64:85348540
Battifora H (1986) The multitumor (sausage) tissue block: novel method for immunohistochemical antibody testing. Lab Invest 55:244248[Medline]
Beckstead JH, Halverson PS, Ries CA, Bainton DF (1981) Enzyme histochemistry and immunohistochemistry on biopsy specimens of pathologic human bone marrow. Blood 57:10881098
Beeckman T, Viane R (2000) Embedding thin plant specimens for oriented sectioning. Biotech Histochem 75:2326[Medline]
Bennett HS, Wyrick AD, Lee SW, McNeil JH (1976) Science and art in preparing tissues embedded in plastic for light microscopy, with special reference to glycol methacrylate, glass knives and simple stains. Stain Technol 51:7197[Medline]
Bradbury AR, Marks JD (2004) Antibodies from phage antibody libraries. J Immunol Methods 290:2949[CrossRef][Medline]
Britten KM, Howarth PH, Roche WR (1993) Immunohistochemistry on resin sections: a comparison of resin embedding techniques for small mucosal biopsies. Biotech Histochem 68:271280[Medline]
Casanova S, Donini U, Zini N, Morelli R, Zucchelli P (1983) Immunohistochemical staining on hydroxyethyl-methacrylate-embedded tissues. J Histochem Cytochem 31:10001004
Casey TT, Cousar JB, Collins RD (1988) A simplified plastic embedding and immunohistologic technique for immunophenotypic analysis of human hematopoietic and lymphoid tissues. Am J Pathol 131:183189[Abstract]
Fejzo MS, Slamon DJ (2001) Frozen tumor tissue microarray technology for analysis of tumor RNA, DNA, and proteins. Am J Pathol 159:16451650
Gerrits P, Horobin R (1996) Glycol methacrylate embedding for light microscopy: basic principles and trouble-shooting. J Histotechnol 19:297311
Glauert AM, Glauert RH, Rogers GE (1956) A new embedding medium for electron microscopy. Nature 178:803
Golick ML, Federman Q (1985) Pressure sensitive adhesive tape for maintaining tissue orientation while embedding in glycol methacrylate. Stain Technol 60:111112[Medline]
Griffiths G, Burke B, Lucocq J (1993) Embedding media for section immunocytochemistry. In Fine Structure Immunocytochemistry. Berlin, Springer-Verlag, 90136
Hand N, Blythe D, Jackson P (1996) Antigen unmasking using microwave heating on formalin fixed tissue embedded in methyl methacrylate. J Cell Pathol 1:3137
Hoos A, Cordon-Cardo C (2001) Tissue microarray profiling of cancer specimens and cell lines: opportunities and limitations. Lab Invest 81:13311338[Medline]
Kerstens HM, Robben JC, Poddighe PJ, Melchers WJ, Boonstra H, de Wilde PC, Macville MV, et al. (2000) AgarCyto: a novel cell-processing method for multiple molecular diagnostic analyses of the uterine cervix. J Histochem Cytochem 48:709718
Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, et al. (1998) Tissue microarrays for high-throughput molecular profiling of tumour specimens. Nat Med 4:844847[CrossRef][Medline]
Kushida H (1959) On an epoxy resin embedding method for ultrathin sectioning. J Electron Microsc (Tokyo) 8:7275
Le Guellec D, Trembleau A, Pechoux C, Gossard F, Morel G (1992) Ultrastructural non-radioactive in situ hybridization of GH mRNA in rat pituitary gland: pre-embedding vs ultra-thin frozen sections vs post-embedding. J Histochem Cytochem 40:979986
Mansy SS (2004) Agarose cell block: innovated technique for the processing of urine cytology for electron microscopy examination. Ultrastruct Pathol 28:1521[Medline]
McCafferty J, Griffiths AD, Winter G, Chiswell DJ (1990) Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348:552554[CrossRef][Medline]
Miyaji T, Hewitt SM, Liotta LA, Star RA (2002) Frozen protein arrays: a new method for arraying and detecting recombinant and native tissue proteins. Proteomics 2:14891493[CrossRef][Medline]
Morey AL, Ferguson DJ, Fleming KA (1995) Combined immunocytochemistry and non-isotopic in situ hybridization for the ultrastructural investigation of human parvovirus B19 infection. Histochem J 27:4653[CrossRef][Medline]
Mozdzen JJ Jr, Keren DF (1982) Detection of immunoglobulin A by immunofluorescence in glycol methacrylate-embedded human colon. J Histochem Cytochem 30:532535
Murray GI, Ewen SW (1991) A novel method for optimum biopsy specimen preservation for histochemical and immunohistochemical analysis. Am J Clin Pathol 95:131136[Medline]
Onetti Muda A, Riminucci M, Bianco P (1992) Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens. Histochemistry 98:283288[CrossRef][Medline]
Shi SR, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741748
Simon R, Mirlacher M, Sauter G (2004) Tissue microarrays. Biotechniques 36:98105[Medline]
Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:3143[CrossRef][Medline]
Tsao-Wu GS, Weber CH, Budgeon LR, Cheng KC (1998) Agarose-embedded tissue arrays for histologic and genetic analysis. Biotechniques 25:614618[Medline]
Wan WH, Fortuna MB, Furmanski P (1987) A rapid and efficient method for testing immunohistochemical reactivity of monoclonal antibodies against multiple tissue samples simultaneously. J Immunol Methods 103:121129[CrossRef][Medline]
Warford A (2004) Tissue microarrays: fast-tracking protein expression at the cellular level. Expert Rev Proteomics 1:283292[CrossRef][Medline]
Westen H, Muck KF, Post L (1981) Enzyme histochemistry on bone marrow sections after embedding in methacrylate at low temperature. Histochemistry 70:95105[CrossRef][Medline]
Widehn S, Kindblom LG (1990) Agarose embedding: a new method for the ultrastructural examination of the in-situ morphology of cell cultures. Ultrastruct Pathol 14:8185[Medline]
Wilson SJ, Wallin A, Della-Cioppa G, Sandstrom T, Holgate ST (2001) Effects of budesonide and formoterol on NF-kappaB, adhesion molecules, and cytokines in asthma. Am J Respir Crit Care Med 164:10471052
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