RAPID COMMUNICATION |
Correspondence to: Moïse Bendayan, Dept. of Anatomy, Université de Montréal, CP 6128 Succ. Centre Ville, Montreal, Quebec H3C 3J7, Canada.
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Summary |
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The biotinyl-tyramide protocol recently introduced for sensitive light microscopic immunocytochemistry was applied to electron microscopy and revealed various tissue antigens with high resolution. The protocol consists of an indirect method in which thin tissue sections are incubated successively within a specific primary antibody, followed by a biotinylated secondary antibody, streptavidin-HRP, and then finally with biotinyl-tyramide. The reaction product appears as a dense filamentous material that is deposited over particular cellular compartments. The labeling obtained for the antigens tested, amylase and heat-shock protein 70 in pancreatic acinar cells, insulin in pancreatic ß-cells, and carbamoyl phosphate synthetase and catalase in liver tissue, was found to be highly specific, with the labeling for each antigen confined to its particular cellular compartment. Background levels and nonspecific deposition of the staining were negligible. The use of biotinyl-tyramide therefore appears to be an alternative sensitive technique for immunoelectron microscopy. (J Histochem Cytochem 45:1449-1454, 1997)
Key Words: immunocytochemistry, electron microscopy, biotinyl-tyramide
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Introduction |
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Since the advent of immunocytochemistry in the late 1940s, major progress has been made in methods of labeling and in techniques of tissue preparation. For electron microscopy, two techniques have been quite successful for demonstration of antigen-antibody complexes: the PAP technique, using peroxidase as the agent of labeling (
Recently, a novel approach for revealing antigen-antibody complexes, the tyramide signal amplification technique, has been reported. First introduced for immunoassays (
In the present study, we report the use of the tyramide signal amplification approach (TSA) for immunoelectron microscopy. Application of the technique on thin sections of tissues and examination at the electron microscopic level have revealed the presence of a dense reaction product at the antigen-antibody reaction sites. This novel technique appears to be simple, reliable, and of high specificity and good resolution. It provides an alternative to existing techniques and could be used in combination with others for multiple staining experiments.
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Materials and Methods |
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Tissue Processing
Small fragments of normal rat pancreatic and liver tissues were fixed by immersion in 0.1 M phosphate-buffered 1% glutaraldehyde for 2 hr at 4C. After fixation, the tissue fragments were washed in the phosphate buffer and processed for low-temperature embedding in Lowicryl as previously described (
Antisera and Reagents
Five different primary antibodies were used to assess the TSA technique: a rabbit anti-human amylase and a mouse monoclonal anti-heat shock protein 70 (HSP-70) from Sigma Chemicals (St Louis, MO), a guinea pig anti-porcine insulin from ICN (Montreal, Quebec, Canada), a rabbit anti-carbamoyl phosphate synthetase (CPS) (
Immunocytochemistry
Labeling was carried out by floating the grids with tissue sections face down on drops of 1% ovalbumin in 10 mM PBS, pH 7.2, for 30 min at room temperature (RT) and then transferring them directly onto a drop of one of the specific primary antibodies for 2 hr at RT or overnight at 4C. The antibodies were used at the following dilutions: anti-amylase 1~10; anti-HSP-70 1~10; anti-insulin 1~200; anti-CPS 1~50; and anti-catalase 1~200. The grids were then rinsed with PBS for 15 min, transferred to the 1% ovalbumin solution for 30 min, and then incubated on a drop of one of the corresponding specific biotinylated secondary antibodies for 60 min at RT: GAR-biotin 1~800 for anti-amylase, anti-CPS, and anti-catalase; GAG-biotin 1~500 for the anti-insulin; and GAM-biotin 1~500 for the anti-HSP-70. Tissue sections were then rinsed with PBS for 15 min, transferred to the ovalbumin solution for 15 min, and then incubated on a drop of streptavidin-HRP diluted 1~500 with PBS for 30 min at RT. The grids were then washed for 15 min with PBS and incubated for 10 min at RT with biotinyl-tyramide diluted 1~50 in 1 x amplification diluent. After washing with PBS and distilled water, the tissue sections were stained with uranyl acetate and examined with a Philips 410SL electron microscope.
The specificity of the labeling with tyramide was assessed by several control experiments: omission of either the primary or secondary antibody and omission of the streptavidin-HRP or the biotinyl-tyramide steps, in four separate experiments.
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Results |
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Application of the TSA technique in combination with specific primary antibodies on thin tissue sections prepared for electron microscopy led to the deposition of a dense filamentous material on structures known to contain the different antigens. The specificity of the labelings appeared to be excellent and is supported by the results obtained using various antigens located in different cells and/or different cell compartments. Incubation of pancreatic tissue with the anti-amylase antibody (Figure 1) led to staining of different compartments of the acinar cells, such as the rough endoplasmic reticulum, the Golgi cisternae, the condensing vacuoles, and the zymogen granules. The dense filamentous reaction product was present on the cell compartments with different intensities, the highest being over the zymogen granules known to contain high amounts of amylase. Other compartments, such as mitochondria or nuclei, were devoid of any staining (Figure 1). The use of the anti-insulin antibody resulted in staining of the pancreatic B-cells, particularly in those compartments involved in secretion, the rough endoplasmic reticulum, the Golgi apparatus, and the immature and mature secretory granules (Figure 2). Staining for HSP-70 was found in the Golgi apparatus and condensing vacuoles of the pancreatic acinar cells and was particularly concentrated in the trans-Golgi network (Figure 3). For the hepatic cells, labeling for carbamoyl phosphate synthetase was restricted to the mitochondria (Figure 4) and that for catalase to the peroxisomes (Figure 5). In all cases, the reaction product appeared as thin filamentous deposits that overlaid the labeled structures without masking their morphological features. Considering the different labeled compartments, the reaction product remained within the limits of each compartment, thus reflecting good resolution. The specificity of the results, as demonstrated by the very low levels of background, was excellent, the labeling being restricted to the corresponding compartments. It was also supported by the control experiments. Omission of the primary or secondary antibodies in the labeling protocol led to absence of staining except for the presence of some random dense spots (Figure 1C). This indicates that application of the TSA protocol without a specific antibody does not generate any staining (Figure 1C). Similar results were obtained when the streptavidin-HRP or the biotinyl-tyramide step was omitted. Nonspecific deposition of the different reagents therefore appears to be negligible. The technique appears reliable and sensitive. However, some precautions must be taken to generate intense and specific stainings. The different solutions should be as fresh as possible, particularly the diluted tyramide solution, which should not be more than 2 weeks old. The use of an old solution affects the chemical reactions and generates labeling of low intensity. Incubation with 1% ovalbumin appears to interfere with the HRP-tyramide reaction and therefore should not be carried out before incubation with the biotinyl-tyramide.
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Discussion |
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The TSA approach was originally introduced for detection of antigens on solid-phase immunoassay (
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Acknowledgments |
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Supported by grants from the Medical Research Council of Canada.
We would like to express our gratitude to Patricia Mayer from NEN Life Science Products (Boston, MA) for her kindness in providing us with the different reagents and her interest in this study.
Received for publication May 8, 1997; accepted July 10, 1997.
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