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 Ca2+ and Mg2+ 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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Yamashita, S. (2016). Post-embedding Mammalian Tissue for Immunoelectron Microscopy: A Standardized Procedure Based on Heat-Induced Antigen Retrieval. In: Schwartzbach, S., Skalli, O., Schikorski, T. (eds) High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol 1474. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6352-2_18
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DOI: https://doi.org/10.1007/978-1-4939-6352-2_18
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Publisher Name: Humana Press, New York, NY
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