Bell P B, Rundquist I, Svensson I, Collins V P
Department of Pathology II, Linköping University, Sweden.
J Histochem Cytochem. 1987 Dec;35(12):1381-8. doi: 10.1177/35.12.3680931.
Immunocytochemistry is routinely used to examine the occurrence and distribution of cytoskeletal proteins in cells, but the results are usually evaluated visually and subjectively. Little use has been made of the potential the method offers for quantitative work. Here we report on application of cytofluorometry to quantify binding of antibodies to the cytoskeleton of U-251 MG human malignant glioma cells in culture. The results show that cytofluorometry is a simple and reliable procedure for: (a) determining the optimal concentrations of primary and secondary antibodies and other labeling reagents; (b) evaluating the binding specificity of commercial secondary antisera; and (c) evaluating the effect of different preparatory procedures on preservation of and binding of antibodies to cytoskeletal structures. Experiments with a monoclonal antibody to tubulin show that preservation of tubulin is very sensitive to the preparatory procedures used. Maximum labeling of tubulin in intact cells was obtained when the cells were pre-fixed with formaldehyde before permeabilization with solvent. Maximum labeling of tubulin in Triton-extracted cytoskeletons was achieved by pre-fixing the cells with the bifunctional protein crosslinking reagent dithiobis (succinimidyl propionate), extracting with Triton in a microtubule-stabilizing buffer, and post-fixing with formaldehyde. GTP was not required to preserve tubulin in cytoskeletons.
免疫细胞化学通常用于检测细胞中细胞骨架蛋白的存在和分布，但结果通常通过视觉主观评估。该方法在定量研究方面的潜力尚未得到充分利用。在此，我们报告了应用细胞荧光测定法对培养的U - 251 MG人恶性胶质瘤细胞的细胞骨架上抗体结合进行定量分析。结果表明，细胞荧光测定法是一种简单可靠的方法，可用于：(a) 确定一抗、二抗和其他标记试剂的最佳浓度；(b) 评估市售二抗血清的结合特异性；(c) 评估不同制备程序对细胞骨架结构上抗体保存和结合的影响。用抗微管蛋白单克隆抗体进行的实验表明，微管蛋白的保存对所用的制备程序非常敏感。在用溶剂通透细胞之前先用甲醛预固定细胞，可使完整细胞中的微管蛋白获得最大标记。在用双功能蛋白交联剂二硫代双（琥珀酰亚胺丙酸酯）预固定细胞、在微管稳定缓冲液中用Triton提取、并用甲醛后固定的情况下，可使Triton提取的细胞骨架中的微管蛋白获得最大标记。在细胞骨架中保存微管蛋白不需要GTP。
