Bell P B, Safiejko-Mroczka B
Department of Zoology, University of Oklahoma, Norman 73019, USA.
Scanning Microsc. 1995 Sep;9(3):843-57; discussion 858-60.
To determine the optimal procedures to preserve cytoskeletal and other macromolecular structures for microscopic studies we have evaluated the effects of various methods to extract cultured cells. In this report, we compare results using different fixatives, crosslinking reagents, and permeabilization methods on (1) the labeling of cells for fluorescence microscopy with phalloidin or antibody against tubulin; and (2) the morphological preservation of macromolecular structures for scanning electron microscopy. Maximal labeling of F-actin with phalloidin was obtained by fixing cells in 4% paraformaldehyde (PFA) and labeling the unextracted cells with methanolic phalloidin, whereas maximal labeling of tubulin required prefixation with either PFA or the bifunctional protein crosslinking reagent, dithiobis (succinimidylpropionate) (DSP) and extraction with ethanol or Triton in a high salt buffer. However, for both qualitative and quantitative light and electron microscopic studies of intracellular macromolecular structures, prefixation with DSP and extracting with Triton X-100 in a stabilizing buffer is the overall method of choice for both labeling and morphological studies. Although other methods provide maximal labeling or preservation of specific structures, this method provides excellent preservation of morphological structure while allowing proteins to be preserved and labeled by specific probes.
为了确定用于显微镜研究的保存细胞骨架和其他大分子结构的最佳方法，我们评估了各种提取培养细胞方法的效果。在本报告中，我们比较了使用不同固定剂、交联试剂和通透化方法对以下两方面的结果：（1）用鬼笔环肽或抗微管蛋白抗体对细胞进行荧光显微镜标记；（2）用于扫描电子显微镜的大分子结构的形态保存。通过将细胞固定在4%多聚甲醛（PFA）中并用甲醇鬼笔环肽标记未提取的细胞，可获得用鬼笔环肽对F-肌动蛋白的最大标记，而微管蛋白的最大标记需要先用PFA或双功能蛋白交联试剂二硫代双（琥珀酰亚胺丙酸酯）（DSP）进行预固定，然后在高盐缓冲液中用乙醇或 Triton 进行提取。然而，对于细胞内大分子结构的定性和定量光镜及电镜研究，在稳定缓冲液中先用DSP预固定并用Triton X-100提取是标记和形态学研究的总体首选方法。尽管其他方法可提供特定结构的最大标记或保存，但该方法在允许蛋白质被特定探针保存和标记的同时，能出色地保存形态结构。
