Severs Nicholas J, Robenek Horst
Imperial College London, National Heart and Lung Division, London, United Kingdom.
Methods Cell Biol. 2008;88:181-204. doi: 10.1016/S0091-679X(08)00411-1.
The term freeze-fracture cytochemistry embraces a series of techniques which share the goal of chemical identification of the structural components viewed in freeze-fracture replicas. As one of the major features of freeze fracture is its ability to provide planar views of membranes, a major emphasis in freeze-fracture cytochemistry is to identify integral membrane proteins, study their spatial organization in the membrane plane, and examine their role in dynamic cellular processes. Effective techniques in freeze-fracture cytochemistry, of wide application in cell biology, are now available. These include fracture-label, label fracture, and the freeze-fracture replica immunolabeling technique (FRIL). In fracture-label, samples are frozen and fractured, thawed for labeling, and finally processed for viewing either by critical-point drying and platinum-carbon replication or by thin-section electron microscopy. Label-fracture involves immunogold labeling a cell suspension, processing as for standard freeze-fracture replication, and then examining the replica without removal of the cellular components. Of greatest versatility, however, is the FRIL technique, in which samples are frozen, fractured, and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate (SDS) to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits the distribution of identified components to be viewed superimposed upon high resolution planar views of replicated membrane structure, for both the plasma membrane and intracellular membranes in cells and tissues. Examples of how these techniques have contributed to our understanding of cardiovascular cell function in health and disease are discussed.
冷冻断裂细胞化学这一术语涵盖了一系列技术，这些技术的共同目标是对冷冻断裂复制品中观察到的结构成分进行化学鉴定。冷冻断裂的一个主要特点是它能够提供膜的平面视图，因此冷冻断裂细胞化学的一个主要重点是鉴定整合膜蛋白，研究它们在膜平面中的空间组织，并检查它们在动态细胞过程中的作用。目前已有在细胞生物学中广泛应用的有效的冷冻断裂细胞化学技术。这些技术包括断裂标记、标记断裂和冷冻断裂复制品免疫标记技术（FRIL）。在断裂标记中，将样品冷冻、断裂，解冻后进行标记，最后通过临界点干燥和铂 - 碳复型或通过超薄切片电子显微镜进行观察处理。标记断裂包括用免疫金标记细胞悬液，按照标准冷冻断裂复型的方法进行处理，然后在不去除细胞成分的情况下检查复制品。然而，通用性最强的是FRIL技术，在该技术中，样品像在标准冷冻断裂中一样进行冷冻、断裂并用铂 - 碳复型，然后用十二烷基硫酸钠（SDS）仔细处理，以去除除附着在复制品本身的一薄层分子之外的所有生物材料。对这些分子进行免疫金标记，可以在细胞和组织中质膜和内膜的复制膜结构的高分辨率平面视图上叠加观察已鉴定成分的分布。本文讨论了这些技术如何有助于我们理解健康和疾病状态下心血管细胞的功能。
