Observations on the kinetics of uranyl acetate and phosphotungstic acid staining of chromatin in thin sections for electron microscopy.

Abstract

When thin sections of spermatogenic chromatin are fixed with either glutaraldehyde alone or postfixed with osmium tetroxide (OsO4) and stained with uranyl acetate (UAc) for increasing times, even after as little as 1 min, stain uptake is proportional to section thickness. Greater UAc uptake is observed in chromatin fixed with glutaraldehyde only, but seen with postfixed chromatin. Lead citrate poststaining of chromatin fixed with either glutaraldehyde or postfixed with OsO4 increases UAc uptake by a factor of about 3. The staining of thin sections of spermatogenic chromatin with ethanolic phosphotungstic acid (PTA) shows a region where stain uptake is proportional to section thickness followed by a plateau. This staining pattern is seen in chromatin fixed with glutaraldehyde alone or postfixed with OsO4; similar levels for final PTA uptake are also observed. An increase in the resin content of embedded chromatin postfixed with OsO4 is proposed to explain the decrease and increase in the rate of migration of UAc and ethanolic PTA staining solutions, respectively.