Immunocytochemical study of fresh unfixed erythrocyte membranes by quick-freezing and deep-etching method

采用快速冷冻和深度蚀刻相结合的方法，对人红细胞膜的细胞质进行了研究。免疫染色的标本被放置在标本支架上，并在液氮中冷却的异戊烷-丙烷混合物(-193ﾟC)中快速冷冻。盖片上的红细胞膜用手术刀在液氮中冷冻断裂，转移到Eiko FD-3AS机中，在-95ﾟC的2-6x10；-7；Torr真空度下深度酸蚀15-30min。然后，通过以24°的角度蒸发铂，然后以90°的角度蒸发碳，制备红细胞膜暴露的细胞质一侧的膜复制品。膜复制品在家用漂白剂中常规处理，并放置在Formvar涂层的铜网上。它们在日立H-8100电子显微镜下进行了检查。电子显微照片是从倒置的底片上打印出来的。在分裂过程中，各种外力引起了膜骨骼组织的变化。最初的变化是膜骨架的外围部分脱落，用单抗抗谱蛋白抗体进行免疫染色。在严重的拉伸条件下，丝状成分的线性重排明显；这些丝状成分平行于红细胞边缘排列，而凹陷的中央部分表现出更紧密的结构。这些变化导致双凹红细胞膜骨架成分在中央硬质区和外周柔软区的分布不同。这表明，抵抗外力的柔韧性区域的可逆膜骨架变化对维持人双凹红细胞的正常结构具有重要意义。

A quick-freezing and deep-etching method in combination with erythrocyte spitting was used to examine the cytoplasmic aspect of whole-mount human erythrocyte membranes. The immunostained specimens were positioned on specimen holders and quickly frozen in an isopentane-propane mixture (-193ﾟC) cooled in liquid nitrogen. The erythrocyte membranes on the coverslips were freeze-fractured in liquid nitrogen with a scalpel, transferred in an Eiko FD-3AS machine and deeply etched at 2-6x10^<-7> Torr vacuum at -95ﾟC for 15-30 min. Membrane replicas of the exposed cytoplasmic side of erythrocyte membranes were then prepared by evaporation of platinum at an angle of 24^O and subsequently of carbon at an angle of 90^O. The membrane replicas were routinely treated in household bleach and placed on Formvar-coated copper grids. They were examined in a Hitachi H-8100 electron microscope. Electron micrographs were printed from inverted negative films. Various external forces induced alterations in membrane skeletal organization during the splitting procedure. The initial change was elogation in the peripheral part of the membrane skeleton, examined by immunostaining with a monoclonal antispectrin antibody. Under severe stretching conditions, a linear rearrangement of filamentous components was evident ; these were disposed parallel to the rim of the erythrocyte, while the central part of the concavity exhibited a more compacted structure. These changes resulted in a different distribution of membrane skeletal components between central rigid and peripheral flexible areas in biconcave erythrocytes. It is suggested that the reversible membrane skeletal changes in the flexible areas which resist the external forces are important for maintaining the normal framework of biconcave human erythrocytes.