ARTICLE |
Correspondence to: Cheryl R. Hann, Mayo Clinic, Guggenheim 920, 200 First Street SW, Rochester, MN 55905. E-mail: hann.cheryl@mayo.edu
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Summary |
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Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.
(J Histochem Cytochem 49:475482, 2001)
Key Words: antigen retrieval, LR White resin, immunolabeling, basement membrane proteins, archival tissue
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Introduction |
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Study of archival tissue provides an opportunity to investigate the pathophysiology of diseases, especially when the archival specimens may represent different stages of a disease process. However, the collection of such specimens usually requires many years, resulting in specimens that have been stored for different lengths of time, often in different fixatives. This can make comparison of labeling for immunoelectron microscopy (IEM) difficult because optimal preservation of tissue antigens requires short fixation times and low concentrations of primary fixative.Antigen retrieval (AR) can recover antigens masked by fixation, storage, processing, and/or resin interactions. It has been used extensively in light microscopy and more recently in IEM (
We studied the basement membrane of Schlemm's canal in the eye, the major route of drainage of aqueous humor from the eye. Alterations in the basement membrane of Schlemm's canal may be important in the pathophysiology of glaucoma (
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Materials and Methods |
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Tissue Samples
Five normal human eyes were removed at autopsy from five individuals (Mayo Institutional Review Board approval 992-99). The mean age was 47.5 ± 33.0 years. None had a history of ocular disease, ocular surgery, or any systemic disease that affected the eye. Causes of death included sepsis, brain death, cardiogenic shock, and motor vehicle accident. Four eyes were from a tissue archive collection and had been fixed in one of four fixatives: 4% paraformaldehyde (4P), 10% buffered neutral formalin (BNF), 4% paraformaldehyde/2% glutaraldehyde (4P/2G), or 1% paraformaldehyde/2% glutaraldehyde (1P/2G), all prepared in phosphate buffer. Storage times for these eyes ranged from 1 month to 9 years (Table 1). Eyes had been stored in fixative at room temperature (RT).
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The fifth eye was obtained unfixed within 18 hr postmortem. It was dissected and portions were placed in four different fixatives. This was done to assess the effect of biological variability on labeling. Tissues from the same eye would be expected to be more similar in labeling than tissues from different individuals. The fixatives were 4P, 10BNF, 4P/2G, and 4% paraformaldehyde/0.1% glutaraldehyde (4P/0.1G). The last fixative did not match any of the fixatives from the eyes above but was chosen because it is often used for IEM fixation. Specimens from each fixative were stored for 3 months before processing and embedding. The 3-month period was chosen to study the effect of longer storage times, although it was a shorter storage period than that of most of the eyes under study.
Immunohistochemical Technique
Wedges of tissue from each eye, containing Schlemm's canal and the trabecular meshwork, were processed for LRWhite embedding using a method modified from
Thin sections (dark gold, 130 nm) were mounted on nickel grids coated with 1.5% parlodion prepared in a 1:1 solution of amyl acetate:acetone. Grids were dipped in this solution and allowed to dry for 30 min before sections were mounted. After sections were mounted, they were allowed to dry overnight on a 50C hotplate.
Multiple grids were made from each tissue block. Some grids were treated with AR before labeling, and others were labeled without AR. AR was done according to the method of
Micrographs were taken of the endothelial cells and underlying basal lamina of Schlemm's canal, trabecular beams with trabecular cells, and adjacent scleral tissue at x25,000 on a JEOL 1200 electron microscope (Peabody, MA). For the four archival specimens, two grids from one tissue block per eye were examined. For the fifth eye, in which comparisons were made within the same eye, one tissue block per quadrant was examined.
Antibodies and Reagents
The antibody to collagen IV was a goat polyclonal antibody to human and bovine Type IV collagen, supplied by Southern Biotechnologies (Birmingham, AL) and used at a 1:100 dilution. The fibronectin antibody was a rabbit polyclonal antibody to human fibronectin obtained from Sigma (St Louis, MO) and was used at a 1:50 dilution. Normal goat serum was obtained from Amersham Life Science (Piscataway, NJ). Secondary antibodies included rabbit anti-goat (5-nm gold particles) and goat anti-rabbit (5-nm gold particles) and were obtained from Amersham Life Science. Both were used at a 1:100 dilution. Silver enhancement was performed with the IntenSE M Silver Enhancement Kit from Amersham Life Science. LR White resin embedding kit was supplied by Ted Pella (Redding, CA). PBSTween, Tris buffer, ovalbumin, and glycine were obtained from Sigma. Dilutions of primary and secondary antibodies were made in PBSTween. Citrate buffer, 0.01 M, pH 6.0, was prepared according to the method of
Quantitation
Quantitation of labeling density was performed to assess changes after AR. For labeling of collagen IV, 10 micrographs at a final magnification of x25,000 were randomly selected from each specimen. A transparent grid measuring 150 mm by 20 mm (3000 mm2; total actual tissue area 4.8 µm2) was overlaid on each micrograph, centered on the basement membrane underlying the cells of Schlemm's canal. Both the inner and outer wall regions were examined. The total number of gold particles present overlying the basement membrane in these regions was counted and the density of labeling was expressed as number of gold particles/µm2 of basement membrane material.
Fibronectin was more diffuse in its labeling pattern than collagen IV, and a larger grid was used (240 mm by 200 mm; each grid square 10 mm x 10 mm, total area 48,000 mm2; total actual tissue area 77 µm2). This grid was placed over the entire region adjacent to Schlemm's canal. On the inner wall, this encompassed the juxtacanalicular tissue, which extends about 10 µm from Schlemm's canal to the first trabecular lamellae. Label on the opposite side of Schlemm's canal, the "outer wall," was also assessed. In contrast to collagen IV, in which label was predominantly in the basement membrane adjacent to the canal, fibronectin label was found in the basement membrane, sheath and tendon material, and other extracellular matrix components. Therefore, all of these were included in the area of possible fibronectin labeling. Fibronectin label occurred as clumps of six to eight gold particles rather than as individual particles as occurred with collagen IV. As a result, fibronectin label was expressed as the number of grid squares containing fibronectin-label/total number of grid squares containing extracellular matrix.
Data are expressed as mean ± SEM. Results were compared using a Student's two-tailed t-test for paired data. T-tests were subjected to the Bonferroni calculation (
Reproducibility of counts was determined by having a second observer count labeling of collagen IV on four specimens, both before and after AR (total of 78 micrographs counted by each).
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Results |
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Collagen IV
Qualitative Evaluation.
Label was found in the basal lamina and adjacent basement membrane material of Schlemm's canal endothelial cells along both the inner wall (IW) and outer wall (OW) of Schlemm's canal (IW, Fig 1A and Fig 1B; OW, Fig 1C and Fig 1D). Label was also observed in the scattered patches of "amorphogranular material" adjacent to the cells in the juxtacanalicular region (
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By subjective assessment, AR increased label for tissue fixed in all four fixatives and for all storage times. AR changed the labeling from sparse patches to a more continuous pattern in the basement membrane (compare Fig 1A and Fig 1B). AR did not cause a new appearance of label in structures that were not labeled on non-AR grids. In tissues fixed in BNF, AR appeared to decrease the granularity and clumping of the label (compare Fig 1B and Fig 1A). A slight amount of nonspecific background labeling was noted with AR over nuclei and in non-tissue areas, such as the lumen of Schlemm's canal.
Quantitative Evaluation. Tissues fixed in 4P/2G had the greatest increase in labeling density, with approximately five times more label than seen in non-AR grids. This increase was most noticeable in the tissue underlying Schlemm's canal (Fig 1G amd 1H). Although this was significant at p=0.02 for this single comparison, it did not reach significance after the Bonferroni correction for multiple comparisons. AR also increased labeling density in tissues fixed in 4P and BNF, but to a lesser extent (Table 1).
Comparison Within the Same Eye. Neither the labeling pattern nor the type of extracellular matrix components that labeled changed with the different fixatives. Although labeling density increased after AR, no "new" components of the extracellular matrix demonstrated label that had not been found in non-AR tissue. Tissue in 4P/2G had the greatest increase in labeling density after AR, with labeling density six times higher than non-AR grids (p<0.01; Table 2). This increase was similar to the increase seen in the four eyes above. Labeling density was increased about twofold after AR for the other fixatives, although this increase was not statistically significant (Table 2).
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Fibronectin
Qualitative Evaluation.
Fibronectin labeling was found adjacent to collagen fibers in the outer wall region of Schlemm's canal (Fig 2A and Fig 2B). In the juxtacanalicular tissue, label was found in the sheath material surrounding the elastic tendons that traverse this region (Fig 2C and Fig 2D). Patches of basement membrane surrounding the loose array of cells in this region were also labeled for fibronectin. Unlike the relatively heavy labeling intensity found with collagen IV, the basement membranes of the inner and outer wall of Schlemm's canal were only sparsely labeled for fibronectin (Fig 2E and Fig 2F). The central core of the trabecular lamellae labeled heavily for fibronectin (Fig 2H), whereas the basement membranes of the trabecular cells lining the lamellae contained minimal label for fibronectin (Fig 2G). The location of labeling was similar in tissues processed with and without AR.
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Fibronectin labeling occurred in aggregated focal clumps rather than in the diffuse pattern of the collagen IV label (Fig 2D and Fig 2F). With AR, the most notable increase in labeling density occurred near collagen fibers in the outer wall of Schlemm's canal (Fig 2A and Fig 2B) and the sheath material surrounding the elastic tendons (Fig 2C and Fig 2D). This was true for all fixatives examined. Label for the central core region of the trabecular lamellae (Fig 2G and Fig 2H) was increased in all fixatives, with the smallest increase noted with 1P/2GL.
Quantitative Evaluation. The greatest increase in labeling density was found for tissue fixed in 4P/2G, which was approximately five times greater than non-AR tissue (p<0.01; Table 1). Label was also increased for other fixatives, with an approximate doubling of label after AR. Tissues fixed in 1P/2G demonstrated the smallest increase after AR.
Comparison Within the Same Eye. The labeling pattern and the type of extracellular matrix components that labeled did not change with the different fixatives nor after AR. Tissue in 4P/2G had the greatest increase in labeling density after AR, with labeling density six times higher than non-AR grids, similar to the increase seen in the four eyes above. Labeling density was increased about twofold after AR for the BNF and 4%P (p<0.01; Table 2).
Control Sections
Sections processed without primary antibody were negative for label for collagen IV and fibronectin. When these negative controls were treated with AR, a small amount of nonspecific background label was present.
Reproducibility
Comparison of results from the two observers revealed that labeling densities were within 12% of each other (78 micrographs counted by both observers).
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Discussion |
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Antigen retrieval increased the labeling of collagen IV and fibronectin of eye tissues after storage in a variety of fixatives and for differing lengths of time. Of importance, although the amount of label increased after antigen retrieval, no additional or "new" extracellular matrix components were labeled after AR. For most fixatives, labeling density generally doubled after AR. A larger increase was seen with 4P/2G, which showed an increase of five to six times that of non-AR grids. Without AR, labeling was best in non-glutaraldehyde-fixed tissue, whereas with AR labeling density increased to become generally similar among fixatives. The exception was 4P/2G, which had a marked increase in labeling. The comparison of fixatives from tissues within the same eye gave similar results. Comparison within the same eye minimizes the effect of biological variability on labeling, which can occur when tissues from different individuals are compared. Overall, these results indicate that the study of archival specimens, often collected in different fixatives over a period of years, is possible with AR methods.
With AR, tissue fixed in 4P/2G had best recovery of both collagen IV and fibronectin labeling. Our findings extend the results of other studies that used AR with low concentrations of fixative for shorter periods of time.
LR White is often employed as the resin of choice to enhance antigenicity. Comparisons of LR White vs Epon-embedded tissues show increased labeling in LR White (
In the current study, tissues fixed in a combination of 4% paraformaldehyde and 2% glutaraldehyde showed a greater increase in label than tissues fixed in lower concentrations of these fixatives. One explanation for this could be the fact that higher-concentration fixatives preserve more epitopes. On dehydration and embedding less washout occurs (
Antigen retrieval can be used with LR White-embedded sections to increase the labeling of the extracellular matrix molecules collagen IV and fibronectin. AR increased the labeling density of tissue in each fixative without changing the overall pattern or distribution of label. Although each antigen is different, AR used with LRWhite is another tool to enable retrospective studies of rare archival tissue specimens.
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Acknowledgments |
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Supported in part by National Institutes of Health research grant EY 07065, Research to Prevent Blindness, Inc. (New York, NY), the Bonner Glaucoma Research Fund (Princeton, NJ), and the Mayo Foundation (Rochester, MN).
Received for publication June 30, 2000; accepted November 15, 2000.
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