Oyamada M, Mori M
J Histochem Cytochem. 1985 Dec;33(12):1197-204. doi: 10.1177/33.12.2415574.
There have been many studies on the localization by immunocytochemistry of cytoskeletal proteins in cells cultured in vitro. However, the distribution of cytoskeleton in cells in situ has yet to be elucidated. In the present study we developed an immunohistochemical method for visualizing tubulin and actin in rat hepatocytes in situ, using a perfusion extraction-fixation procedure, in which the liver was perfused through the portal vein with a nonionic detergent to make the plasma membranes permeable to soluble substances, followed by a fixative to preserve cytoskeletal structure. Using the immunogold and peroxidase-antiperoxidase (PAP) staining procedures, we found that in hepatocytes in situ, tubulin was localized in cytoplasmic filamentous networks and in spindle fibers, as in hepatocytes and other cells in vitro. On the other hand, the distribution of actin in hepatocytes in situ was considerably different from that in well-spread hepatocytes and other cells cultured in vitro. In hepatocytes in situ, actin did not form any stress fibers, but was distributed preferentially under the plasma membrane, especially around the bile canaliculi. The perfusion extraction-fixation procedure could be adapted to visualize cytoskeleton in other tissues.
关于体外培养细胞中细胞骨架蛋白的免疫细胞化学定位，已有许多研究。然而，细胞原位细胞骨架的分布仍有待阐明。在本研究中，我们开发了一种免疫组织化学方法，用于原位观察大鼠肝细胞中的微管蛋白和肌动蛋白，采用灌注提取 - 固定程序，即通过门静脉用非离子去污剂灌注肝脏，使质膜对可溶性物质具有通透性，然后用固定剂保存细胞骨架结构。使用免疫金和过氧化物酶 - 抗过氧化物酶（PAP）染色程序，我们发现，原位肝细胞中的微管蛋白定位于细胞质丝状网络和纺锤体纤维中，这与体外培养的肝细胞和其他细胞中的情况相同。另一方面，原位肝细胞中肌动蛋白的分布与铺展良好的肝细胞和体外培养的其他细胞中的分布有很大不同。在原位肝细胞中，肌动蛋白不形成任何应力纤维，而是优先分布在质膜下方，尤其是胆小管周围。灌注提取 - 固定程序可适用于观察其他组织中的细胞骨架。
