An ultrastructural study of the cytoplasmic aspects of erythrocyte membranes by a quick-freezing and deep-etching...

A novel method for preparing exposed cytoplasmic aspects of erythrocyte membranes is described which improves the resolution in direct electron microscope images. Normal human erythrocytes were briefly fixed with paraformaldehyde and pelleted. Glass coverslips were coated with 3-aminopropyl triethoxysilane and glutaraldehyde to make erythrocytes stick to them. A drop containing the erythrocyte pellets was sandwiched between 2 coverslips. The attached erythrocytes were split open mechanically and postfixed with glutaraldehyde. Some were treated with Triton X-100 or postfixed with osmium tetroxide. All specimens were quickly frozen in an isopentane-propane mixture, deeply etched and rotary shadowed with platinum and carbon. Filamentous structures were seen to form networks on the cytoplasmic sides of the erythrocyte membranes. Triton X-100 and osmium tetroxide fixation distorted the fine network structure. This quick-freezing and deep-etching method will be useful in the analysis of the in situ ultrastructure of the cytoplasmic sides of erythrocyte membranes.