Safiejko-Mroczka B, Bell P B
Department of Zoology, The University of Oklahoma, Norman, USA.
J Histochem Cytochem. 1996 Jun;44(6):641-56. doi: 10.1177/44.6.8666749.
Because permeabilization of the cell membrane is necessary to label intracellular proteins with most fluorescent probes, it is important to optimize the preservation and labeling of the proteins under study. We used qualitative and quantitative fluorescence microscopy to evaluate the effects of six different bifunctional protein cross-linking reagents and several extraction conditions on the labeling of filamentous actin with phalloidin and the immunolabeling of tubulin and gelsolin. The labeling of cytoskeletal and associated proteins can be significantly enhanced by the appropriate combination of bifunctional protein cross-linking reagents and extraction conditions. However, the conditions that give the most intense labeling vary depending on the label used. The greatest intensity of labeling with either phalloidin or antibodies was obtained with the intermediate-length cross-linker DSP. The two-step procedure of cross-linking with DSP and extracting in Triton X-100 in microtubule-stabilizing buffer containing DSP gives maximal labeling with phalloidin. Maximal labeling of gelsolin and tubulin with antibodies is obtained by extracting DSP-cross-linked cells with Triton in Hank's saline containing DSP. Therefore, DSP reproducibly improves preservation of both soluble and filamentous proteins for quantitative and qualitative studies by fluorescence microscopy.
由于使用大多数荧光探针标记细胞内蛋白质时细胞膜通透化是必要的，因此优化所研究蛋白质的保存和标记非常重要。我们使用定性和定量荧光显微镜来评估六种不同的双功能蛋白质交联试剂以及几种提取条件对用鬼笔环肽标记丝状肌动蛋白、微管蛋白和凝溶胶蛋白免疫标记的影响。双功能蛋白质交联试剂和提取条件的适当组合可显著增强细胞骨架及相关蛋白质的标记。然而，产生最强标记的条件因所用标记而异。使用中等长度交联剂DSP可获得用鬼笔环肽或抗体标记的最大强度。在含有DSP的微管稳定缓冲液中用DSP交联并在Triton X-100中提取的两步法可使鬼笔环肽标记达到最大值。通过在含有DSP的汉克氏盐溶液中用Triton提取DSP交联的细胞，可获得用抗体对凝溶胶蛋白和微管蛋白的最大标记。因此，DSP可重复性地改善可溶性和丝状蛋白质的保存，用于荧光显微镜的定量和定性研究。
