Temkin R J, So D Y, Lea P J
Canadian Genetic Diseases Network, Faculty of Medicine, University of Toronto, Ontario.
Microsc Res Tech. 1993 Oct 15;26(3):260-71. doi: 10.1002/jemt.1070260308.
Kidneys of anesthetized rats were perfused with digitonin to extract cytosolic proteins of glomerular podocytes so that the remaining intracellular structures could be examined by three-dimensional stereo high-resolution scanning electron microscopy (HRSEM). Cytoskeleton, consisting of microtubules and intermediate filaments, was preserved with each applied concentration of digitonin. High concentrations of digitonin (1.0 mg/ml) produced a corrugated appearance in plasma membranes likely due to the formation of digitonin-cholesterol complexes. At 1.0 mg/ml digitonin, the Golgi complex became vesicularized, and mitochondria were well extracted and their ultrastructure preserved. Lower concentrations of digitonin (0.1 and 0.2 mg/ml) were less disruptive to both the plasma membrane and the Golgi complex. Mitochondria, rough endoplasmic reticulum, coated vesicles, nuclear membrane, and chromatin were well preserved. Extraction with digitonin, at the optimal concentration and perfusion time, simultaneously maintains both the cytoskeleton and membranous organelles inside the cell and provides a method to elucidate the interactions between these two components. Furthermore, digitonin extraction should preserve antigenic sites, thereby allowing the localization of intracellular proteins by backscattered electron imaging of immunogold labels in the scanning electron microscope.
用洋地黄皂苷灌注麻醉大鼠的肾脏，以提取肾小球足细胞的胞质蛋白，以便通过三维立体高分辨率扫描电子显微镜（HRSEM）检查剩余的细胞内结构。由微管和中间丝组成的细胞骨架在每个应用浓度的洋地黄皂苷下均得以保留。高浓度的洋地黄皂苷（1.0 mg/ml）使质膜呈现出波纹状外观，这可能是由于洋地黄皂苷 - 胆固醇复合物的形成所致。在1.0 mg/ml洋地黄皂苷浓度下，高尔基体变成囊泡状，线粒体被充分提取且其超微结构得以保留。较低浓度的洋地黄皂苷（0.1和0.2 mg/ml）对质膜和高尔基体的破坏较小。线粒体、粗面内质网、被膜小泡、核膜和染色质均保存良好。在最佳浓度和灌注时间下用洋地黄皂苷进行提取，可同时维持细胞内的细胞骨架和膜性细胞器，并提供一种阐明这两种成分之间相互作用的方法。此外，洋地黄皂苷提取应保留抗原位点，从而通过扫描电子显微镜中免疫金标记的背散射电子成像来定位细胞内蛋白质。
