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Resin Tissue Microarrays : a Universal Format for Immunohistochemistry

William J. Howat, Anthony Warford, Joanne N. Mitchell, Kay F. Clarke, Jen S. Conquer and John McCafferty

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

	Summary

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Tissue microarray (TMA) technology allows the miniaturization and characterization of multiple tissue samples on a single slide and commonly uses formalin-fixed paraffin-embedded (FFPE) tissue or acetone-fixed frozen tissue. The former provides good morphology but can compromise antigenicity, whereas the latter provides compromised morphology with good antigenicity. Here, we report the development of TMAs in glycol methacrylate resin, which combine the advantages of both methods in one embedding format. Freshly collected tissue fixed in –20C acetone or 10% neutral buffered formaldehyde were cored and arrayed into an intermediary medium of 2% agarose before infiltration of the agarose array with glycol methacrylate resin. Acetone-fixed resin TMA demonstrated improved morphology over acetone-fixed frozen TMA, with no loss of antigenicity. Staining for extracellular, cell surface, and nuclear antigens could be realized with monoclonal and polyclonal antibodies as well as with monomeric single-chain Fv preparations. In addition, when compared with FFPE TMA, formalin-fixed tissue in a resin TMA gave enhanced morphology and subcellular detail. Therefore, resin provides a universal format for the construction of TMAs, providing improved tissue morphology while retaining antigenicity, allows thin-section preparation, and could be used to replace preparation of frozen and FFPE TMAs for freshly collected tissue. (J Histochem Cytochem 53:1189–1197, 2005)

Key Words: tissue microarray • resin TMA • glycol methacrylate • immunohistochemistry • single-chain Fv

	Introduction

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THE TERM "TISSUE MICROARRAYS" (TMAs) was coined in 1998 with the description of the arraying of multiple (1000) cores of tissue in one recipient paraffin wax block (Kononen et al. 1998). Although not the first attempt at increasing throughput of tissue samples for simultaneous analysis (Battifora 1986; Wan et al. 1987), the provision of instrumentation for easy construction of arrays has led to an increase in the use of TMAs in a variety of settings. TMAs are typically constructed by the removal of 0.6-mm to 2-mm diameter cores of tissue from donor samples and transferal of these to a recipient block in a grid-like fashion, thus retaining a link to the original block and its pathology (Kononen et al. 1998). For optimal preparation, areas of appropriate morphology or antigenicity are selected from the original donor blocks and cores taken from those areas, thus ensuring inclusion of relevant and representative tissue (Simon et al. 2004). Most TMA studies have been undertaken using archival formalin-fixed paraffin-embedded (FFPE) tissue; however, the recent preparation of frozen TMAs has extended the TMA application to areas where antigenicity is not compromised through formalin fixation (Fejzo and Slamon 2001; Hoos and Cordon-Cardo 2001). The preparation of frozen arrays is similar to that of paraffin arrays except that to avoid ice crystal artifact, all coring and preparation must be performed at subzero temperatures. Although greater preservation of antigen can be achieved using frozen sections, an inherent problem in standard methods is that morphology is inferior to that of equivalent FFPE preparations.

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 radical–mediated 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.

	Materials and Methods

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Tissue Collection and Fixation
C57BL6/J-Tyr^C-Brd albino homozygotes were housed in specific pathogen–free facilities in accordance with the Home Office Code of Practice for the Housing and Care of Animals used in Scientific Procedures on a 12-hr daylight/night cycle. All animals were housed in individual ventilated cages supplied by Tecniplast UK Ltd. Animals were killed according to the Home Office Schedule 1 guidelines. All tissues, except for female sexual organs, were collected from male animals. Tissues collected included adrenal, bladder, cerebellum, cerebrum, large intestine, small intestine, kidney, liver, lung, cardiac muscle, striated muscle, esophagus, ovary, skin, spleen, stomach, testis, thymus, trachea, and uterus.

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 40–45C. Sections were picked up on SuperFrost Plus slides and dried at 40–45C 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-house–generated 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).

	Results

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Tissue Microarray Construction
To enable the construction of TMAs in resin, an intermediary holding medium had to be produced that would give structure to the TMA yet allow infiltration and polymerization of the GMA monomer. Following the method of Kerstens et al. (2000) and further experimentation with different concentrations of agarose, a 2% agarose gel was chosen. Although providing good support, this did not allow removal of an agarose core; therefore, the 0.6-mm donor needle and stylet was used to disperse rather than core the agarose.

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 400–500 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.

View larger version (155K): [in this window] [in a new window]   Figures 1 and 2 Figure 1 Image of 20-tissue resin TMA taken by stereo microscopy (A); arrows indicate cores that have skewed toward the end of the array. Hematoxylin staining of resin section of trachea (B) and colon (C). c, cartilage; e, epithelium. Bars = 20 µm. Figure 2 Micrographs comparing acetone-fixed resin TMA (A,C) and acetone-fixed frozen TMA (B,D) following immunohistochemistry. CD45 (A,B) and laminin (C,D) demonstrated. Bars = 20 µm.

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 2A–2D). 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 3A–3C). 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).

View larger version (182K): [in this window] [in a new window]   Figures 3 and 4 Figure 3 Micrographs of TMA sections following immunohistochemistry. Kidney stained with laminin from 2-µm resin TMA section (A), 7-µm frozen TMA section (B), and 4-µm paraffin TMA section (C). Glomeruli are indicated by an arrow in each micrograph. Bars = 50 µm. Figure 4 Micrographs of 2-µm resin TMA sections. Acetone-fixed muscle tissue stained with single-chain Fv to desmin using tyramine amplification (A) and control (B). Formalin-fixed kidney stained with laminin (C) and spleen stained with Ki-67 (D). Bars = 20 µm.

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.

	Discussion

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Resin embedding and thin-sectioning methods have long been in use in electron microscopy for resolution of fine structural detail. The more common epoxy resins used in electron microscopy provided hardness for thin sectioning, but owing to their hydrophobic nature and heating during polymerization, they have hindered histochemical staining. In contrast, acrylic resins, such as glycol methacrylate (2-hydroxyethyl methacrylate, GMA) and other acrylate derivatives, have been used as embedding media for light and electron microscopy (Bennett et al. 1976; Griffiths et al. 1993). Their water-miscible nature has allowed their use in both enzyme histochemistry and IHC (Casanova et al. 1983; Onetti Muda et al. 1992), but their use has been limited by the formaldehyde fixation regime employed. Several alternative techniques, such as the use of methylmethacrylate for IHC following formaldehyde fixation (Hand et al. 1996) and freeze-substitution (Murray and Ewen 1991), have provided improved immunohistochemistry profiles. However, the former method does not allow the use of fixation-sensitive antibodies and the latter would not be suitable for a re-arraying process, inasmuch as the infiltration and embedding process is conducted at subzero temperatures. In contrast, a –20C acetone fixation/dehydration step, combined with incubation and processing with methyl benzoate, provided the opportunity to use antibodies previously designated as suitable for "frozen only" (Casey et al. 1988; Britten et al. 1993).

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. coli–based 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 direction—across 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.

	Acknowledgments

 
The authors would like to acknowledge Dr. Susan Wilson (Histochemistry Research Unit, University of Southampton) and Dr. K. Steel (Team 27, Genetics of Deafness, Wellcome Trust Sanger Institute) for their advice; Cambridge Antibody Technology Ltd. for their provision of the antibody library that was selected to generate the anti-desmin scFv clone; and its production by the other members of the Atlas of Protein Expression team at the Sanger Institute.

	Footnotes

 
Received for publication February 21, 2005; accepted May 16, 2005

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