Ultrastructural localization of nonheme cellular iron with ferrocyanide

Abstract

The Prussian blue reaction was evaluated at the ultrastructural level as a cytochemical method to identify ferric and ferrous iron in rat bone marrow and splenic macrophages. Satisfactory tissue preservation and staining were achieved after fixation for 1 hr in 3% glutaraldehyde and exposure for 30 min to Perls's ferrocyanide solution before routine osmication and embedding. The acid ferrocyanide solution formed cuboidal and irregular electron-opaque deposits which localized ferric iron in the macrophage siderosomes and hyaloplasm. When thin sections were directly stained with the acid ferrocyanide, the stain deposits were much less distinct. The size and number of precipitates varies from one phagosome to another. Recently phagocytosed erythrocytes exhibited sparse evenly distributed stain deposits. Several cells displayed abundant precipitates on the inner surface of the plasmalemma. Prussian blue precipitates were occasionally seen in mitochondria and nuclear euchromatin. Although osmium tetroxide post-fixation improved tissue preservation, it did not enhance the density of the ferri-ferrocyanide precipitate. The ferrocyanide solution yielded cuboidal deposits also in clots impreganted with ferritin, and electron diffraction analysis confirmed the symmetrical crystal structure of these stain precipitates. Smaller irregular precipitates were formed in clots impregnated with FeCl3, or Fe2(SO4)3 solutions, despite the equally intense blue coloring of these specimens at the light microscope level. This result was interpreted as indicating that the iron hydroxide core or protein structure of ferritin and hemosiderin contributed to the formation of the ultrastructurally evident cuboidal precipitates, but were not necessary for the formation of a colored reaction product. The acid ferrocyanide solution failed to stain clots formed in FeCl2, CuCl2 or CuCl solutions. Staining with a ferricyanide solution identified only spare foci of ferrous iron in some siderosomes. This study demonstrates that the Prussian blue reaction can be used ultrastructurally to localize iron cations bound to some nonheme iron binding proteins.