Lemanski L F, Paulson D J, Hill C S, Davis L A, Riles L C, Lim S S
J Histochem Cytochem. 1985 Jun;33(6):515-22. doi: 10.1177/33.6.3889138.
A procedure has been developed for the immunoelectron microscopic localization of intracellular antigens on thin-sectioned tissues. The tissues were fixed in a periodate-lysine-paraformaldehyde solution or a formaldehyde-glutaraldehyde combination and embedded in the acrylate-methacrylate mixture, Lowicryl K4M (Polaron), which was polymerized under ultraviolet irradiation at -35 degrees C. Thin sections were mounted on gold grids, immunostained using an indirect method with ferritin-labeled antibodies, and, optionally, counterstained with osmium tetroxide and/or lead citrate and uranyl acetate. The procedure provided good morphologic preservation of the cell architecture in adult and embryonic heart, and skeletal and smooth muscle tissue, as well as nonmuscle cells. At the same time it retained the antigenicities of several contractile proteins, including myosin, tropomyosin, actin, and alpha-actinin. The method has advantages over en bloc staining techniques in that the problem of antibody penetration into the cells is eliminated and careful controls can be performed on adjacent sections. This technique will be useful for localizing, at the ultrastructural level, contractile and other selected proteins in a variety of muscle and non-muscle cells. Details of the new protocol and a description of the results of using antibody against the contractile protein, alpha-actinin, are given.
已开发出一种用于在薄切片组织上进行细胞内抗原免疫电子显微镜定位的方法。组织用高碘酸盐-赖氨酸-多聚甲醛溶液或甲醛-戊二醛组合固定，然后包埋在丙烯酸酯-甲基丙烯酸酯混合物Lowicryl K4M（宝灵曼公司）中，该混合物在-35℃紫外线照射下聚合。将薄切片安装在金网上，使用铁蛋白标记抗体的间接方法进行免疫染色，并且可选择地用四氧化锇和/或柠檬酸铅及醋酸铀进行复染。该方法能很好地保存成年和胚胎心脏、骨骼肌和平滑肌组织以及非肌肉细胞的细胞结构形态。同时，它保留了几种收缩蛋白的抗原性，包括肌球蛋白、原肌球蛋白、肌动蛋白和α-辅肌动蛋白。该方法相对于整体染色技术具有优势，因为消除了抗体渗透到细胞中的问题，并且可以在相邻切片上进行仔细对照。该技术将有助于在超微结构水平上定位各种肌肉和非肌肉细胞中的收缩蛋白及其他选定蛋白。给出了新方案的详细信息以及使用针对收缩蛋白α-辅肌动蛋白的抗体的结果描述。
