Electron staining of the cell surface coat by osmium-low ferrocyanide

Abstract

In aldehyde-fixed liver and renal cortex of rat and mouse several variations of postfixation with osmium tetroxide plus potassium ferrocyanide ( FeII ) were tried. Depending on the ferrocyanide concentration different staining patterns were observed in TEM. -Osmium-High Ferrocyanide [40 mM (approximately 1%) OsO4 + 36 mM (approximately 1.5%) FeII , pH 10.4], stains membranes and glycogen. Cytoplasmic ground substance, mitochondrial matrices and chromatin are partially extracted, cell surface coats remain unstained. Membrane contrast, but extraction too, are higher with solutions containing cacodylate- than phosphate-buffer. -Osmium-Low Ferrocyanide [40 mM (approximately 1%) OsO4 + 2 mM (approximately 0.08%) FeII , pH 7.4], stains cell surface coats and basal laminae, but not glycogen, except for special cases. The trilaminar structure of membranes is poorly delineated. Signs of cytoplasmic extraction are not visible. The surface coat staining is stronger and more widespread with solutions containing phosphate- instead of cacodylate-buffer; it is enhanced by section staining with lead citrate. The cell surface coat stain does not traverse tight junctions nor permeate membranes.