Membrane skeleton in fresh unfixed erythrocytes as revealed by a rapid-freezing and deep-etching method

A rapid-freezing and deep-etching method for examining en face the cytoplasmic aspects of unfixed erythrocyte membranes is described, which provides improved resolution. Normal human erythrocytes were centrifuged, washed in a phosphate buffer solution and pelleted. Glass coverslips were coated with 3-aminopropyl triethoxy silane and glutaraldehyde to make erythrocytes stick to them. A drop containing the erythrocyte pellet was sandwiched between 2 coverslips. The attached erythrocytes were slowly split open in the cytosol buffer solution. The specimens on coverslips were rapidly frozen in an isopentane-propane mixture (-193 degrees C), deeply etched and rotary shadowed with platinum and carbon. Filamentous structures were observed to form fine networks on the cytoplasmic side of erythrocyte membranes. The length of the filaments was shorter than that previously reported for glutaraldehyde-fixed filaments. The number of intersections between filaments was increased as compared with the previous data. It is concluded that dense in situ networks of short filaments beneath erythrocyte membranes can be viewed in a relatively intact state by splitting fresh unfixed specimens followed by the rapid-freezing and deep-etching method.