VISUALIZATION OF IRON IN CULTURED MACROPHAGES - A CYTOCHEMICAL LIGHT AND ELECTRON-MICROSCOPIC STUDY USING AUTOMETALLOGRAPHY

The objective of this study was to develop a sensitive cytochemical me thod for the visualization of iron, both at light microscopical (LM) a nd at electron microscopical (EM) levels, in glutaraldehyde-fixed cult ured cells with reasonable morphological preservation. The method is b ased on autometallography (also called the sulfide silver method or th e Timm technique). Gold, silver. and various metal sulfides have previ ously been shown to act as catalysts for cellular silver deposition fr om a physical developer (autometallography). In our modification of th is cytochemistry, a high pH is used during the initial sulfidation ste p to guarantee adequate levels of sulfide ions to generate enough Fe(I i or III) sulfide. Since this procedure may cause severe cellular dist ortion. we initially stabilize the cultured cells by a glutaraldehyde fixation. We have compared our new high pH, high S2- LM and EM variety of autometallography with other modifications of this technique that have previously been used for LM and EM demonstration of easily sulfid ated heavy metals, such as zinc. Cultured mouse macrophages were exami ned for the localization of reactive metals following endocytosis of f erritin or inorganic Fe(III) iron. Ag-precipitates, presumed to indica te the presence of iron. were predominantly found within secondary lys osomes of the acidic vacuolar apparatus. The relation of the Ag-precip itates 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 lysos omal content-and a few extralysosomal sites-of reactive substances. be lieved to he iron.