pH-dependent effect of heat-induced antigen retrieval of epoxy section for electron microscopy |
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| Authors: | Brorson S-H |
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| Affiliation: | 1. A*STAR, Bioinformatics Institute, Singapore, Singapore;2. ECE, University of Alberta, Edmonton, AB, Canada;3. Zhejiang University, Hangzhou, China;4. Beijing Institute of Technology, Beijing, China;5. A*STAR, Institute for Infocomm Research, Singapore, Singapore;1. Department of Veterinary Surgery, Nippon Veterinary and Life Science University, 1-7-1 Kyonancho, Musashino, Tokyo 180-8602, Japan;2. Department of Veterinary Anatomy, Nippon Veterinary and Life Science University, 1-7-1 Kyonancho, Musashino, Tokyo 180-8602, Japan;1. Institute of Molecular Genetics, Biosafety Research and Consulting, Johannes Gutenberg-University, Becher-Weg 30a, 55128 Mainz, Germany;2. Institute of Evolution, University of Haifa, Mount Carmel, Haifa 31905, Israel |
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| Abstract: | The aim was to examine how the pH in the antigen retrieval medium (citrate) affects the yield of immunogold labeling of epoxy sections. Renal swine tissue with glomerular immune complex deposits with reactivity against IgG was embedded in epoxy resin. Prior to immunogold labeling with anti-IgG, ultrathin sections from these blocks were exposed to antigen retrieval by heating in citrate solution (pH 6, 9 or 12) at 95 degrees C in a PCR-machine or at 121 or 135 degrees C min in an autoclave. The level of immunogold labeling was significantly higher for pH 12 than for pH 6 when heated at 95 degrees C (50% more intense), but at the cost of the ultrastructural preservation of the tissue. At pH 12 and temperature 135 degrees C the epoxy sections were completely destroyed. The sections which had been heated at 135 degrees C, pH 6 appeared significantly better both with respect to intensity of immunogold labeling (85% more intense) and to ultrastructural preservation than those which were heated at 95 degrees C, pH 12. Therefore, our results indicate that relatively low pH (pH 6) and high temperature is the method of choice, but low temperature and high pH can be used when an autoclave is not available. |
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