ARTICLE |
Correspondence to: Christoph Röcken, Inst. of Pathology, Otto-von-Guericke-University, Leipziger Str. 44, D-39120 Magdeburg, Germany.
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Summary |
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The advantages of using immunoelectron microscopy in amyloid research and surgical pathology for the classification of amyloid deposits are well documented. The aim of this study was to improve single-labeling postembedding immunostaining by testing different antigen retrieval (AR) techniques. Etching and AR procedures were applied to sections from aldehyde-fixed and Epon-embedded autopsy specimens of patients who had suffered from generalized AA amyloidosis, systemic senile ATTR amyloidosis, or generalized -light chain amyloidosis. The procedures used were no AR, H2O2, saturated aqueous sodium metaperiodate (mPJ), heating in deionized water (dH2O), heating in sodium citrate buffer (SCB), heating in EDTA (each 91C, 30 min), and combinations of etching and heating. Little effect was evident after treatment with H2O2, mPJ, and heating in dH2O, but the signal density markedly increased after heating in 1 mM EDTA. Heating in SCB affected immunolabeling with anti-transthyretin and anti-
-light chain, whereas no effect was achieved for immunolabeling with anti-AA amyloid. We concluded that AR may significantly improve immunostaining of specimens that have undergone conventional fixation and embedding procedures for electron microscopy. The effect of AR on the detection of amyloid fibril proteins was probably mediated in part through chelation or binding of metal ions by the AR medium. (J Histochem Cytochem 47:13851394, 1999)
Key Words: amyloid, antigen retrieval, immunoelectron microscopy
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Introduction |
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Amyloidoses are characterized by local organ-limited or generalized proteinaceous deposits of autologous origin (
The diagnosis and classification of amyloidosis require histological evidence using either light or electron microscopy, both of which have an identical specificity (
Immunotyping of amyloid presents several problems. First, the amyloid fibril proteins may differ from their autologous precursors in their tertiary structure resulting from amyloid formation (- and
-light chain origin (
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Materials and Methods |
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Tissues
Specimens were obtained from three autopsy cases. The first was a 64-year-old white man who had suffered from rheumatoid arthritis; specimens from heart, lung, liver, kidney, spleen, and thyroid were fixed overnight in 5% neutralized formalin. The second case, an 82-year-old white man, had suffered from cholecystitis due to cholecystolithiasis; specimens from heart, lung, liver, kidney, spleen, pancreas, duodenum, and prostate were fixed and stored in 5% neutralized formalin, either overnight (paraffin embedding) or for 7 weeks (Epon embedding) before processing. The third case was a 72-year-old white man who had suffered from a plasmacytoma; specimens from heart, lung, liver, and kidney were fixed and stored in 5% formalin for 5 weeks before processing. The autopsies had been performed 1, 4, and 2 days after death, respectively. For light microscopy, specimens were embedded in paraffin. Deparaffinized sections were stained with hematoxylin and eosin. The presence of amyloid was demonstrated by the appearance of green birefringence from alkaline alcoholic Congo Red staining under polarized light (-light chain, and
-light chain, as described elsewhere (
Electron Microscopy
For electron microscopy, the autopsy specimens were fixed in different ways. Specimens from the second and third case were postfixed in a mixture of 2% formalin/2.5% glutaraldehyde (pH 7.2, overnight, 4C) and subsequently in 3.125% glutaraldehyde only (7 hr, 4C). Specimens from the first case were fixed in three different ways: Procedure 1, initial fixation in 5% formalin [pH 7.0, overnight, room temperature (RT) followed by 3.125% glutaraldehyde (pH 7.2, 16 hr, 4C); Procedure 2, initial fixation in a mixture of 2% formalin/2.5% glutaraldehyde (pH 7.2, overnight, 4C) and subsequently in 3.125% glutaraldehyde (pH 7.2, overnight, 4C); or Procedure 3, in a mixture of 4% paraformaldehyde/0.2% glutaraldehyde (pH 7.2, 40 hr, 4C) only. After the different fixation procedures, all samples were washed in PBS, postfixed in 2% OsO4Veronal buffer [2% OsO4 in 2.94 g 5,5-sodium barbital, 1.94 g sodium acetate, 500 ml deionized water (dH2O); pH 7.2, 4 hr, 4C], and rinsed thoroughly in Veronal buffer (three times for 5 min, 4C) and dH2O (three times for 5 min, RT). Aqueous uranyl acetate 3% (60 min, RT) was then added and the specimens were dehydrated stepwise in 20, 30, and 50% ethanol (10 min each, RT). After overnight incubation in 70% ethanol (4C), the specimens were treated with 1% uranyl acetate in 70% ethanol (60 min, RT) and further dehydrated in 96% (twice for 30 min, RT) and absolute ethanol (three times for 30 min, RT). Before embedding the samples were incubated in propylene oxide (twice for 15 min, RT) and propylene oxide 1:1 Epon (51 ml Epon 812, 37 ml methylnadic anhydride, 12 ml dodecenyl succinic anhydride; 3 hr, RT). Finally, the specimens were embedded in Epon supplemented with 2% DMP-30 and polymerization took place over 24 hr at 60C. Semithin sections (1 µm) were stained with Toluidine blue. Ultrathin sections (120 nm) were mounted on copper grids and counterstained with 3% aqueous uranyl acetate (30 min, RT), then contrasted with 1% aqueous lead citrate (15 min, RT).
Etching and Antigen Retrieval Procedures
Postembedding immunostaining of ultrathin sections was performed after the following etching and AR procedures were applied: (a) no treatment; (b) etching in H2O2; (c) treatment with saturated aqueous sodium metaperiodate (mPJ); (d) heating in dH2O; (e) heating in sodium citrate buffer (SCB); (f) heating in EDTA; (g) etching in H2O2 followed by heating in dH2O; (h) etching in H2O2 followed by heating in SCB; (i) etching in H2O2 followed by heating in EDTA; (j) treatment with mPJ followed by heating in dH2O; (k) treatment with mPJ followed by heating in SCB; (l) treatment with mPJ followed by heating in EDTA.
For postembedding immunoelectron microscopy, ultrathin sections (120 nm) were mounted on Formvar-coated nickel grids (200 mesh; Plano, Wetzlar, Germany). Etching and treatment were carried out using 10-µl drops of either 3% H2O2 (10 min, RT) or mPJ solution (60 min, RT) on Parafilm sheets in microwell culture plates, using a moist chamber for the latter. Antigen retrieval (AR) was performed using either dH2O (30 min, 91C), 10 mM SCB (10 ml 0.1 M citric acid monohydrate, 90 ml 0.1 M trisodium citrate dihydrate, and 900 ml dH2O; pH 6.0, 30 min, 91C), or 1 mM EDTA (pH 8.0, 30 min, 91C). One mM EDTA was prepared using a stock solution of 500 mM EDTA adjusted to pH 8.0 with NaOH. A working solution was obtained by diluting the stock solution 1:500 with dH2O. For AR, the following heating set-up was applied. Using a scalpel or a razor blade, slim slots were cut along the longitudinal axis of one end of a stiff plastic tube measuring 1012 cm in length and 6 mm external diameter. The grids were tucked mechanically into the slots. The elastic tension of the tubing was strong enough to maintain the position of the grids during heating, and no glue was required. A cork was attached to the opposite end of the tubing and the whole assembly was put into a test tube filled with the preheated AR solution and placed in a hot water bath. The temperature was controlled by two thermometers. One was placed inside a control test tube without grids and the other in the surrounding water bath. The test tubes were supported by a 4-well plastic rack (see Figure 1 and Figure 2).
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Postembedding Immunolabeling
Postembedding immunolabeling was carried out after etching and AR. For immunolabeling with anti-AA amyloid and anti--light chain, the grids were rinsed in 50 mM Tris-buffered saline (Tris-HCl; pH 7.4, three time for 5 min, RT) and incubated in Tris-HCl containing 5% (w/v) fetal calf serum (Tris-FCS; 30 min, RT). Tris-FCS was removed without washing the specimens and the primary antibodies were applied, i.e., anti-AA amyloid (dilution 1:100; 1 hr at RT; monoclonal), or anti-
-light chain (1:167; 1 hr at RT; polyclonal; both from DAKO, Carpinteria, CA). Immunostaining was visualized by the appropriate 15-nm colloidal gold-labeled secondary antibodies (EM 15-nm rabbit anti-mouse IgG and EM 15-nm goat anti-rabbit IgG; both from Biotrend, Cologne, Germany; 1:20, 60 min, RT). Between the incubations the specimens were washed with Tris-FCS (five times for 5 min). The antibodies were then crosslinked with 1% glutaraldehyde in Tris-HCl (pH 7.4, 15 min, RT). The immunolabeling procedure with anti-transthyretin (1:50; overnight at 4C; polyclonal; DAKO) was identical except that PBS, pH 7.4, was used instead of Tris-HCl. Finally, the sections were counterstained with 3% aqueous uranyl acetate (30 min, RT) and contrasted with 1% aqueous lead citrate (15 min, RT). Fixation, staining, and contrasting were each followed by an extensive washing in dH2O. The specificity of immunostaining was controlled by omitting the primary antibodies. The sections were air-dried and inspected in a Zeiss EM900 electron microscope.
Quantitation of Probe Density
The mean numerical probe density was quantified as follows. In each experiment, 10 pictures were taken (at a magnification of x20,000) at random from amyloidotic areas. This was performed in triplicate and the number of gold particles was counted in the whole frame of 30 pictures. The mean number of gold particles per frame was calculated. At a magnification of x20,000, the individual frame covered 13.68 µm2. As a control, the number of gold particles in nonamyloidotic areas was quantified in the same manner. Statistics were performed using the V2.04a GraphPad Instat program.
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Results |
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Light Microscopy
In the first and third autopsy cases, a variable amount of vascular and interstitial amyloid deposit was found in every organ investigated (Figure 3 and Figure 7).
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In the first case, immunohistochemistry showed intense staining of the amyloid deposits with the antibody directed against AA amyloid only (Figure 4), and the diagnosis was a generalized AA amyloidosis secondary to rheumatoid arthritis. The patient had died from renal insufficiency caused by amyloid deposition.
In the third case, immunohistochemical classification of the amyloid deposits showed immunostaining with the antibody directed against -light chain only (Figure 8), and the diagnosis was a generalized
-light-chain amyloidosis secondary to a plasmacytoma. The patient had died from cardiac failure caused by massive cardiac and pulmonary deposition of amyloid.
In the second case, a variable amount of vascular amyloid deposit was found only in the left and right cardiac ventricles, lung, liver, duodenum, and prostate (Figure 5). No amyloid was evident in the kidneys, spleen, and pancreas. The deposits were immunoreactive for transthyretin only (Figure 6) and were interpreted as systemic senile amyloidosis. The patient had died from cardiac failure after cholecystectomy, which was not attributable to amyloid deposition.
Electron Microscopy
Electron microscopic examination of the specimens from all cases showed irregular arrangements of linear, nonbranching fibrils with an indefinite length and an approximate diameter of 1012 nm. These features are typical of amyloid.
Immunoelectron Microscopy
Specimen Selection.
For immunoelectron microscopic investigation of the first and third cases, specimens were sought with abundant vascular and interstitial deposits of amyloid, interspersed with large areas without amyloid, the latter serving as an internal negative control. In the first case, specimens from the thyroid only proved useful because massive deposits of amyloid encircled follicles homogeneously filled with colloid. For the same reason, specimens from the lung were selected in the third case, in which massive deposits of amyloid encircled optically empty alveolar spaces. In the second case, specimens from the heart only enclosed a sufficient amount of vascular amyloid deposits appropriate for immunoelectron microscopic investigation.
Postembedding Immunolabeling
Controls.
Background staining of postembedding immunolabeling was investigated by omitting the primary antibody. Formvar-coated nickel grids yielded a weak background staining with gold particles, whereas no particles were found on blank ultrathin sections of Epon. Irrespective of whether etching or AR was performed, tissue sections from the thyroid, heart, and lung yielded no or a negligible amount, of gold particles when the primary antibody was omitted (Figure 11). Application of primary antibodies against AA amyloid on sections from the heart (ATTR amyloidosis) and lung (A amyloidosis) and against
-light-chain on sections from the thyroid (AA amyloidosis) and heart (ATTR amyloidosis) showed only weak nonspecific labeling of the specimens and no signal enhancement was found on amyloid deposits. Immunostaining of the lung (A
amyloidosis) and thyroid (AA amyloidosis) with anti-transthyretin showed intense nonspecific labeling without signal enhancement on amyloid.
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Specimen Preservation After Etching and Antigen Retrieval
Treatment with mPJ significantly reduced the contrast of the sections but the amyloid fibrils were still discernible. Etching with H2O2 occasionally affected the specimens, resulting in reduced contrast and loss of tissue structure. After AR using SCB or EDTA, a variable number of different sized "holes" were found, although the integrity of the ultrathin section and the contrast appeared to be maintained. To further analyze the holes, AR was tested on ultrathin sections mounted on nickel grids without Formvar coating. No holes were evident, and we concluded that they were artifacts attributible to the use of Formvar.
First Case (AA Amyloidosis)
Postembedding immunolabeling of ultrathin sections from the thyroid with the antibody directed against AA amyloid yielded immunostaining immediately related spatially to amyloid fibrils. The mean density of gold particles was 31.1/13.68 µm2 (Table 1). Except for heating in EDTA (82.3/13.68 µm2), no other etching, pretreatment, or AR procedure significantly improved immunolabeling with anti-AA amyloid (Table 1). Background staining was only moderately influenced by AR (8.0/13.68 µm2 after EDTA retrieval vs 1.8/13.68 µm2 without specimen pretreatment) and did not account for increased immunolabeling. Heating in dH2O and etching in H2O2 followed by heating in SCB significantly reduced immunostaining (Table 1).
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To investigate the influence of fixation on AR, three different aldehyde fixation procedures were tested [i.e., initial fixation in 5% formalin followed by 3.125% glutaraldehyde (Procedure 1), initial fixation in a mixture of 2% formalin/2.5% glutaraldehyde and subsequently in 3.125% glutaraldehyde (Procedure 2), and fixation in a mixture of 4% paraformaldehyde/0.2% glutaraldehyde only (Procedure 3)]. These had little effect on the intensity of immunostaining enhanced by EDTA pretreatment.
Second case (ATTR Amyloidosis)
Postembedding immunolabeling of ultrathin sections from the heart with an antibody against transthyretin showed immunostaining immediately related spatially to amyloid fibrils (Figure 9 and Figure 10). The mean density of gold particles was 103.3/13.68 µm2 (Table 2). Except for a combined pretreatment with H2O2 and EDTA (306.9/13.68 µm2) and mPJ and EDTA (319.1/13.68 µm2), no other etching, pretreatment, or AR procedure significantly improved immunolabeling with anti-transthyretin (Figure 9 and Figure 10; Table 2). However, background staining in EDTA-treated specimens increased significantly after etching with H2O2 (57.4/13.68 µm2) and mPJ (93.7/13.68 µm2) compared to that found with no etching (24.5/13.68 µm2). Therefore, an increased immunolabeling of amyloid was interpreted as being due to an overall increase in the background staining. H2O2 (10.6/13.68 µm2) and mPJ (19.0/13.68 µm2) alone without subsequent EDTA treatment did not increase the background staining compared to that found with no treatment (20.1/13.68 µm2).
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Third Case (A Amyloidosis)
Postembedding immunolabeling of ultrathin sections from the lung with an antibody against -light chain yielded immunostaining immediately related spatially to the amyloid fibrils (Figure 12 Figure 13 Figure 14). The mean density of gold particles was 67.0/13.68 µm2. Specimen pretreatment with either H2O2, mPJ, or hot dH2O had no significant effect on immunolabeling, as indicated by the mean number of gold particles per frame (see Table 3). A significant increase was achieved after the specimens had been treated either with SCB or EDTA. EDTA showed the most significant effect on immunolabeling, yielding a mean number of 241.1/13.68 µm2 (Figure 13 and Figure 14; Table 3). Furthermore, the effect of EDTA was reduced after etching with H2O2. Neither pretreatment with mPJ nor etching with H2O2 had any significant effect on immunolabeling. The mean numbers of gold particles on nonamyloidotic tissue areas were 3.8/13.68 µm2 (no treatment), 2.5/13.68 µm2 (mPJ + SCB), 34.0/13.68 µm2 (no etching + EDTA), and 20.3/13.68 µm2 (mPJ + EDTA). Therefore, the moderately increased background staining had a negligible effect on the overall enhanced immunolabeling of the amyloid fibrils.
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Discussion |
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Heat-mediated antigen retrieval (AR) is a nonenzymatic pretreatment for immunohistochemical staining that is widely used in research and surgical pathology (for reviews see
Several different methods can be used to achieve AR, all of which probably reflect the different but ill-defined and unpredictable biochemical mechanisms of AR (
Aldehyde fixatives, such as formalin and glutaraldehyde, are crosslinkers that are preferred for tissue fixation because they are superior for preservation of tissue structure for both light and electron microscopy (
In this study we applied such a test battery of different etching and AR procedures to improve the postembedding immunohistochemical classification of amyloid in electron microscopic specimens that were fixed and embedded by conventional procedures, i.e., formalin and glutaraldehyde fixation and embedding in Epon. Our results demonstrate that, for these specimens, AR shows antigen/antibody-specific retrieval patterns. Immunolabeling with a monoclonal antibody against AA amyloid was sensitive only to EDTA retrieval, and no other specimen pretreatment had a significant effect. Immunostaining with anti-transthyretin was sensitive to EDTA and to the combinations of H2O2/EDTA and mPJ/EDTA. However, enhanced signal density after the latter two AR procedures was due to a dramatically increased background staining, which was not observed for the other two antigens/antibodies under investigation. The possibility that increased background staining may have been due to increased nonspecific binding of the secondary antibody (
Unlike anti-AA and anti-transthyretin, immunolabeling with anti--light chain was improved by a combined pretreatment with mPJ/SCB and mPJ/EDTA, and was not merely due to increased background staining. However, it is interesting that none of the antigens/antibodies investigated showed improved staining after heating in dH2O. Therefore, it is likely that the AR was mediated not by heat and rehydration alone but by the presence of a chelating or metal binding agent, such as EDTA or SCB (
In immunoelectron microscopy, etching is applied either to increase the surface area of Epon, and thereby the number of antigens available for antibody binding, or to reverse the negative effect of osmium tetroxide fixation on antigen preservation (-light chain amyloidosis: mPJ + SCB), enhancing background staining (ATTR amyloidosis), or counteracting the effect of AR. AR using only EDTA improved immunostaining with anti-AA, but this result was abolished when EDTA was combined with etching in H2O2 or mPJ. Similar observations were made with
-light chain amyloidosis (H2O2 + EDTA). Therefore, the effect of etching in combination with AR is unpredictable and must be assessed by trial and error using a test battery.
Different methods have been described for the application of heat for antigen unmasking in immunoelectron microscopy. These include microwaving, autoclaving, pressure cooking, and boiling in an open beaker (
In summary, our study shows that AR may significantly improve immunostaining of specimens that have undergone conventional fixation and embedding procedures for electron microscopy. In our series of tests, the effect of AR on the detection of amyloid fibril proteins was probably mediated in part through chelation or binding of metal ions by the AR medium. The application of a test battery proved valuable in assessing the appropriate etching and AR procedure, because the effect of etching and AR is unpredictable for a given antigen/antibody.
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Acknowledgments |
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We thank Ms Becher, Ms Drueg, Ms Koçalkova, and Ms Röder for excellent and skillful assistance. The specimens from the third autopsy case were kindly provided by Professor Dr. Med. Otto (Dortmund, Germany).
Received for publication March 2, 1999; accepted May 18, 1999.
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