Severs N J
Department of Cardiac Medicine, National Heart and Lung Institute, London U.K.
J Microsc. 1991 Jan;161(Pt 1):109-34. doi: 10.1111/j.1365-2818.1991.tb03077.x.
A wide variety of methods by which cytochemistry and freeze-fracture can be successfully combined have recently become available. All these techniques are designed to provide information on the chemical nature of structural components revealed by freeze-fracture, but differ in how this is achieved, in precisely what type of information is obtained, and in which types of specimen can be studied. Colloidal gold labelling is the most widely used cytochemical technique in freeze-fracture cytochemistry, and for many of the methods it is indispensable. In principle, there are four points in which the cytochemical labelling step may be integrated into the standard freeze-fracture procedure: (i) before the specimen has been frozen, (ii) after it has been fractured and thawed, (iii) after platinum shadowing or (iv) after completion of the full replication sequence. Retention of the gold label so that it can be viewed with replicas can be achieved by depositing platinum and/or carbon upon the labelled surface, thereby partially entrapping the marker particles within the replica, or by retaining, attached to the replica, fragments of fractured membrane (or other cellular components) that would normally have been lost during the replica cleaning step. Another approach to visualizing the label is to use sections, either with portions of a replica included face-on, or for examining the fracture path through the sample (without replica). Recent developments have centered on the use of replicas to stabilize half-membrane leaflets; not only may these and associated attached components be retained for labelling just before mounting, but they provide a means for manipulating the specimen--specifically, turning it over during processing--so that additional structural information can be obtained. This article aims to explain how modern freeze-fracture cytochemistry works, and how the various techniques differ in what they can tell us about membranes and other cellular structures. With the effectiveness of many of the techniques now demonstrated, freeze-fracture cytochemistry is firmly established, alongside a range of related labelling techniques, for increasing application in cell and membrane biology in the 1990s.
最近出现了各种各样能成功将细胞化学与冷冻断裂相结合的方法。所有这些技术旨在提供有关冷冻断裂所揭示的结构成分化学性质的信息，但在实现方式、获取的具体信息类型以及可研究的标本类型方面存在差异。胶体金标记是冷冻断裂细胞化学中使用最广泛的细胞化学技术，并且在许多方法中它是不可或缺的。原则上，细胞化学标记步骤可在四个点整合到标准冷冻断裂程序中：（i）在标本冷冻之前；（ii）在标本断裂和解冻之后；（iii）在铂阴影投射之后；或（iv）在完整复制序列完成之后。通过在标记表面沉积铂和/或碳，从而将标记颗粒部分截留在复制品内，或者通过保留附着在复制品上的断裂膜（或其他细胞成分）碎片（这些碎片在复制品清洗步骤中通常会丢失），可以实现金标记的保留以便能用复制品观察到。另一种观察标记的方法是使用切片，要么包含正面朝上的复制品部分，要么用于检查穿过样品的断裂路径（不使用复制品）。最近的进展集中在使用复制品来稳定半膜小叶；这些以及相关的附着成分不仅可以在安装前保留用于标记，而且它们提供了一种操纵标本的方法——具体来说，在处理过程中翻转标本——以便可以获得额外的结构信息。本文旨在解释现代冷冻断裂细胞化学的工作原理，以及各种技术在它们能告诉我们有关膜和其他细胞结构的信息方面有何不同。随着现在许多技术的有效性得到证明，冷冻断裂细胞化学与一系列相关标记技术一起，在20世纪90年代细胞和膜生物学中的应用日益广泛，已牢固确立。
