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 0504, ferrocyanide-reduced 0504, osmiumthiocarbohydrazide-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

Quantitative ultrastructural analysis of perifibrous lipid and its association with elastin in nonatherosclerotic human aorta.

“…Varying thiocarbohydrazide incubation time to 2 and 10 minutes did not change the results appreciably. Use of potassium ferrocyanide in combination with OsO 4 89 or as a part of the OTO procedure 5 offered little advantage in staining lipid droplets or in structure identification for our purposes. Routine en bloc processing was performed with some samples by using 2% aqueous OsO 4 for 1 hour, washing three times in 0.05 M Na maleate buffer (pH 5.2), staining in 1 % uranyl acetate in maleate buffer (pH 6.0) for 1 hour, and repeat washing in maleate buffer (pH 5.2).…”

“…5 Preliminary work showed that tissue penetration by this procedure was limited to a depth of approximately 150 micrometers. Therefore, following primary fixation, all tissue slices were further sectioned at 100 to 200 micrometers using a Vibratome Tissue Sectioner.…”

“…The preferred method (OTO) for secondary fixation and staining en bloc was as follows: incubation for 30 minutes in 2% aqueous OsO 4 , extensive washing with four buffer changes of 3 minutes each, a brief wash in distilled water, incubation in 1.5% aqueous thiocarbohydrazide (Polysciences) for 5 minutes, washing with buffer and water, and repeated OsO 4 incubation for 30 minutes. 5 ' 7 The thiocarbohydrazide solution was freshly prepared by intermittent shaking for 3 to 4 hours at 58° C, cooling to 25°C…”

“…The poor retention and staining of neutral lipid by routine techniques has been the chief deterrent to adequate ultrastructural description of perifibrous lipid. In the course of attempting to enhance lipid identification by several methods, we became aware of a report by Willingham and Rutherford 5 that neutral and membranous lipid in tissue culture cells could be stained for electron microscopy by an osmium-thiocarbohydrazide-osmium (OTO) sequence. They had adapted the technique from an earlier one by Seligman and colleagues.…”

Practical Solutions to Frequent Problems Encountered in Thin Sections Electron Microscopy

“…Some groups opt out of using uranyl acetate altogether and instead add potassium ferricyanide to the osmium solution: this reduces OsO 4 and stains structures with metallic osmium, but without producing "dots" [4][5][6]. It is, however, not often appreciated that uranyl acetate and potassium ferricyanide have different properties.…”