McDonald Kent L, Sharp David J, Rickoll Wayne
Cold Spring Harb Protoc. 2012 Oct 1;2012(10):1044-8. doi: 10.1101/pdb.top068452.
There is no single, simple procedure for fixing and embedding all tissues for transmission electron microscopy (TEM). The chemistry of different cell types is to some extent unique, and this affects the way each cell type reacts to the wide array of fixatives, buffers, organic solvents, and resins used in TEM specimen preparation. A recurring theme in those organisms or cell types that are difficult to fix is the presence of a diffusion barrier that prevents the free diffusion of fixative and other chemicals in and out of the cell or tissue. This in turn means that fixation takes a relatively long time (measured in minutes or tens of minutes in some cases), during which the cells begin autolysis or are otherwise degraded from their original state. Drosophila requires specific preparation methods for TEM because most fly tissues are surrounded by significant diffusion barriers. In the embryo, it is the vitelline envelope, and in larvae and adults, it is the cuticle. In this article, we discuss methods that have evolved to cope with these barriers to achieve reasonable preservation of ultrastructure.
对于透射电子显微镜（TEM）样本制备而言，不存在一种简单统一的方法来固定和包埋所有组织。不同细胞类型的化学组成在一定程度上是独特的，这会影响每种细胞类型对TEM样本制备中所使用的各种固定剂、缓冲液、有机溶剂和树脂的反应方式。在难以固定的生物体或细胞类型中，一个反复出现的问题是存在扩散屏障，该屏障会阻止固定剂和其他化学物质自由进出细胞或组织。这进而意味着固定需要相对较长的时间（在某些情况下以分钟或数十分钟来衡量），在此期间细胞开始自溶或以其他方式从原始状态降解。果蝇的TEM样本制备需要特定的方法，因为大多数果蝇组织都被显著的扩散屏障所包围。在胚胎中，是卵黄膜；在幼虫和成虫中，则是表皮。在本文中，我们将讨论为应对这些屏障而发展出的方法，以实现对超微结构的合理保存。
