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 20 mM Tris–HCl buffer (pH 9.0) for 1 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. (2010). The Post-embedding Method for Immunoelectron Microscopy of Mammalian Tissues: A Standardized Procedure Based on Heat-Induced Antigen Retrieval. In: Schwartzbach, S., Osafune, T. (eds) Immunoelectron Microscopy. Methods in Molecular Biology, vol 657. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60761-783-9_19
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DOI: https://doi.org/10.1007/978-1-60761-783-9_19
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Publisher Name: Humana Press, Totowa, NJ
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