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

点击翻译按钮获取中文摘要

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.