Chapter title |
Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen Retrieval.
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Chapter number | 18 |
Book title |
High-Resolution Imaging of Cellular Proteins
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Published in |
Methods in molecular biology, January 2016
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DOI | 10.1007/978-1-4939-6352-2_18 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6350-8, 978-1-4939-6352-2
|
Authors |
Shuji Yamashita |
Editors |
Steven D. Schwartzbach, Omar Skalli, Thomas Schikorski |
Abstract |
We describe a standardized method of fixation, antigen retrieval, and image contrasting for post-embedding immunoelectron microscopy. Tissues are fixed with formaldehyde solutions containing Ca(2+) and Mg(2+) ions at pH 7.4 and then at pH 8.5. After dehydration with dimethylformamide, the specimens are embedded in LR-White resin. For antigen retrieval, ultrathin sections are heated in 0.5 M Tris-HCl, pH 9.0, for 1-2 h at 95 °C. After immunogold labeling, the sections are treated with a mixture of tannic acid and glutaraldehyde, with OsO4 solution, and then double-stained with uranyl acetate and lead citrate. The standardized method yields strong and reproducible immunoreactions for many antigens showing excellent image contrast without destruction of fine structures. |
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