Negative staining-carbon film technique: new cellular and molecular applications

Two techniques are presented which extend the original negative staining-carbon film technique into new areas of cellular and molecular application. These relate (1) to the production of negatively stained specimens of single-layer plasma membrane split from intact cells during the overall procedure that are negatively stained from the cytoplasmic face and (2) to the production of negatively stained specimens directly from glycerol-containing protein solutions, membrane or viral suspensions. In both cases in vacuo drying onto mica from glycerol is performed, prior to deposition of a carbon film. (For the cellular technique, freshly cleaved mica is firstly rendered positively charged by immersion in Alcian blue.) This is followed by release of the carbon film plus adsorbed membrane or protein by floating onto water, with subsequent negative staining. Selected preliminary applications using human erythrocyte membrane and the high molecular weight (native) human erythrocyte tripeptidyl peptidase-II complex are given and considered speculation as to the future application of the techniques is provided.