Visualization of iron in cultured macrophages: A cytochemical light and electron microscopic study using autometallography

Abstract

The objective of this study was to develop a sensitive cytochemical method for the visualization of iron, both at light microscopical (LM) and at electron microscopical (EM) levels, in glutaraldehyde-fixed cultured cells with reasonable morphological preservation. The method is based on autometallography (also called the sulfide silver method or the Timm technique). Gold, silver, and various metal sulfides have previously been shown to act as catalysts for cellular silver deposition from a physical developer (autometallography). In our modification of this cytochemistry, a high pH is used during the initial sulfidation step to guarantee adequate levels of sulfide ions to generate enough Fe(II or III) sulfide. Since this procedure may cause severe cellular disrortion, we initially stabilize the cultured cells by a glutaraldehyde fixation. We have compared our new high pH, high S 2− LM and EM variety of autometallography with other modifications of this technique that have previously been used for LM and EM demonstration of easily sulfidated heavy metals, such as zinc. Cultured mouse macrophages were examined for the localization of reactive metals following endocytosis of ferritin or inorganic Fe(III) iron. Ag-precipitates, presumed to indicate the presence of iron, were predominantly found within secondary lysosomes of the acidic vacuolar apparatus. The relation of the Ag-precipitates to iron was proven by the fact that iron-exposed cells showed a much reduced amount of silver precipitates after subsequent exposure to deferoxamine a potent iron chelator. Moreover, control macrophages neither exposed to iron nor to ferritin showed only a low normal lysosomal content—and a few extralysosomal sites—of reactive substances, believed to be iron.