Use of osmium tetroxide-potassium ferricyanide in reconstructing cells from serial ultrathin sections

Abstract

We describe a technique, modified from Langford and Coggeshall [Anat. Rec., 197 (1980) 297-303; J. Comp. Neurol., 203 (1981) 745-750], for enhancing membrane contrast and defining cellular boundaries, that is useful for reconstructing individual cells from serial ultrathin sections. The cells of interest in our study were neuronal germinal cells and their differentiated progeny in the retinas of young goldfish. These cells were labeled by pulse injections of [3H]thymidine, and they were subsequently identified in EM autoradiographs by the presence of silver grains overlying their nuclei. In tissue prepared by traditional procedures (fixation in mixed aldehydes, postfixation in osmium tetroxide) it was difficult to follow the processes of these cells through the complex, dense network of cells in the differentiated retina. However, in tissue postfixed with a mixture of osmium tetroxide and potassium ferricyanide, the contrast of the cell membranes was improved and, in favorable preparations, a dense precipitate was formed in the extracellular spaces, serving to outline individual cells. This greatly faciliated the preparation of reconstructions from serial ultrathin sections.