The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells

Abstract

The preservation and contrast of membranous structures in cultured cells using various postfixation procedures prior to embedding have been investigated. These include routine OsO4, ferrocyanide-reduced OsO4, osmium-thiocarbohydrazide-osmium (OTO), and ferrocyanide-reduced osmium-thiocarbohydrazide-ferrocyanide-reduced osmium (R-OTO). With standard ethanol-Epon dehydration/embedding techniques, a dramatic improvement in both membrane contrast and preservation of bilayer membrane structure was achieved using preembedding OTO in cultured cells. R-OTO yielded similar enhanced preservation and contrast of membranes. Both of these methods also resulted in an increase in the contrast of diaminobenzidine reaction product from horseradish peroxidase activity, and of lipid droplets and lipoprotein particles. However, R-OTO did not cause the same increase in the density of proteinaceous elements as was seen with the OTO method. Ferrocyanide-reduced osmium alone showed significant advantages for quantitation of immunocytochemistry using ferritin labels with bismuth subnitrate counterstain. These methods should have general usefulness for the preservation of lipid-containing structures in cultured cells.