Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples
Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples
复制标题
DOI：
10.1046/j.1365-2818.2003.01228.x
复制
发表时间：
2003-10-01
影响因子：
2
通讯作者：
Giddings, TH
中科院分区：
文献类型：
--
作者：
Giddings, TH
基于高压冷冻和冷冻替代的标本制备方法使电子显微镜生物样品的形态保存质量取得了显着进步。然而，可视化的一个子集的细胞膜，特别是内质网和顺高尔基体，往往是受损的缺乏对比。相比之下，增加膜染色的一些努力可能导致过度的颗粒染色。目前还没有一种冷冻置换法既能克服这些局限性，又能适用于所有类型的分析。然而，在大多数情况下，以下一个或多个协议，也许有微小的修改，应该产生令人满意的结果。在戊二醛和乙酸铀酰丙酮中冷冻取代，然后包埋在Lowicryl HM 20中，产生适合免疫定位和高分辨率结构研究的样品。膜通常是轻度染色，但非常明确。在接触四氧化锇之前，在丙酮中的单宁酸和戊二醛中的初始冷冻置换显著增强了哺乳动物细胞膜上的对比度。最后，初步试验表明，在丙酮中的高锰酸钾冷冻置换可以提供内质网和高尔基体以及其他膜的强烈对比。高锰酸盐在室温下用于水溶液时降解细胞骨架元素和其他蛋白质的趋势在用作冷冻置换试剂时明显减少。
Specimen preparation methods based on high-pressure freezing and freeze-substitution have enabled significant advances in the quality of morphological preservation of biological samples for electron microscopy. However, visualization of a subset of cellular membranes, particularly the endoplasmic reticulum and cis Golgi, is often impaired by a lack of contrast. By contrast, some efforts to increase membrane staining may lead to excessively granular staining. No one freeze-substitution method has emerged that both overcomes these limitations and is suitable for all types of analysis. However, one or more of the following protocols, perhaps with minor modifications, should yield satisfactory results in most cases. Freeze-substitution in glutaraldehyde and uranyl acetate in acetone, followed by embedding in Lowicryl HM20, generates samples suitable for both immunolocalization and high-resolution structural studies. Membranes are typically lightly stained but very well defined. Initial freeze-substitution in tannic acid and glutaraldehyde in acetone prior to exposure to osmium tetroxide significantly enhanced contrast on mammalian cellular membranes. Finally, initial trials indicate that freeze-substitution in potassium permanganate in acetone can provide strong contrast on endoplasmic reticulum and Golgi as well as other membranes. The tendency of permanganate to degrade cytoskeletal elements and other proteins when employed in aqueous solutions at room temperature is apparently curtailed when it is used as a freeze-substitution reagent.