A Controlled Silver Impregnation Method to Characterize Cultured Cardiomyocytes

Abstract

A morphological characterization of cultured cardiomyocytes was attempted using a modification of a silver impregnation technique originally described for connective tissue. Cardiac cells, obtained from newborn rats and grown as dissociated cultures on plastic surfaces, were fixed in methanol plus 5% glacial acetic acid, treated with potassium permanganate, decolorized in oxalic acid, sensitized with potassium bichromate, impregnated with a silver-ammonium complex, reduced in gelatin-formalin preparation, toned with gold chloride and fixed in sodium thiosulfate. The cultured cardiac cells tended to form a monolayer, although many myocytes remained isolated. Spherical nuclei, sharply stained with silver, were centrally located and surrounded by relatively plentiful cytoplasm packed with well delineated myofibrils. Contaminating fibroblasts were readily distinguished by their spindle-shaped nuclei and the presence of overstained collagen fibers, as well as the absence of myofibrils. In the absence of specific antibody for immunocytochemical identification of cardiomyocytes, morphological characterization of cell type and degree of differentiation by the controlled silver impregnation procedure described here provides a viable alternative, both in short- and long-term studies.