Bleher R, Kandela I, Meyer D A, Albrecht R M
Department of Animal Science, University of Wisconsin, 1675 Observatory Drive, Madison, WI 53706, USA.
J Microsc. 2008 Jun;230(Pt 3):388-95. doi: 10.1111/j.1365-2818.2008.01997.x.
Multiple-labelling immuno-EM is a powerful tool for localizing and co-localizing different antigens simultaneously in cells and tissues at high spatial resolution. Commonly used labels for this purpose are differently sized gold spheres. A comparison of results obtained with differently sized markers is often difficult, because the diameters of markers influence labelling efficiency. In the current study, we investigate a method for high-resolution multiple-labelling immuno-EM, using equally sized colloidal markers made of different metals. Energy filtering transmission electron microscopy is used to differentiate particles based on elemental composition. The labels consist of colloidal gold, palladium and platinum-core gold-shell particles of approximately 6 nm in diameter, which are conjugated to different primary antibodies. Applicability of the electron spectroscopic imaging, methodology is demonstrated by labelling of actin, alpha-actinin and myosin on ultra-thin cryosections of skeletal muscle tissue.
多重标记免疫电子显微镜是一种强大的工具，可在细胞和组织中以高空间分辨率同时定位和共定位不同抗原。为此常用的标记物是不同大小的金颗粒。比较不同大小标记物获得的结果往往很困难，因为标记物的直径会影响标记效率。在本研究中，我们研究了一种高分辨率多重标记免疫电子显微镜方法，使用由不同金属制成的大小相同的胶体标记物。能量过滤透射电子显微镜用于根据元素组成区分颗粒。标记物由直径约6nm的胶体金、钯和铂核金壳颗粒组成，它们与不同的一抗结合。通过对骨骼肌组织超薄冷冻切片上的肌动蛋白、α-辅肌动蛋白和肌球蛋白进行标记，证明了电子光谱成像方法的适用性。
