The presence of ferric and ferrous iron in the nonheme iron store of resident macrophages in different tissues and organs: histochemical demonstrations by the perfusion-Perls and -Turnbull methods in the rat.

We previously developed the highly sensitive perfusion-Perls and -Turnbull methods to visualize nonheme ferric (Fe (III)) and ferrous (Fe (II)) iron, respectively. The present study used these methods to investigate the possible presence of nonheme iron in the redox (ferric/ferrous) state in the noneheme iron store (phagolysosomes and siderosomes) of resident macrophages in the rat. The perfusion-Perls and -Turnbull methods at pH 0.6 supplemented by DAB intensification intensely stained resident macrophages of different tissues and organs of normal and iron-overloaded rats. The perfusion-Turnbull method, which is specific for nonheme Fe (II), partly stained hemosiderin at pH 5.3, but hardly stained it at the physiological pH, suggesting the presence of some iron in the reduced form, free Fe2+ and/or loosely bound Fe (II), at the intravacuolar pH (5.4+/-0.2) of the phagolysosomes of macrophages. Electron microscopy of the splenic and hepatic macrophages treated by the perfusion-Perls or -Turnbull method showed that Fe (II) deposits were largely distributed along the margin of hemosiderin masses while Fe (III) deposits could also be found within hemosiderin masses.

Perfusion-Perls and -Turnbull methods supplemented by DAB intensification for nonheme iron histochemistry: demonstration of the superior sensitivity of the methods in the liver, spleen, and stomach of the rat.

Perfusion-Perls and -Turnbull methods supplemented by the intensification with 3,3'-diaminobenzidine (+ DAB) enabled stronger and more extensive staining of nonheme iron than the Perls + and Turnbull + DAB methods carried out on tissue sections fixed with 10% formalin in 0.9% saline or PBS. The section- and perfusion-Perls + DAB methods are not specific for the demonstration of nonheme ferric iron but also stain nonheme ferrous iron. However, owing to its high sensitivity, the perfusion-Perls + DAB method would provide useful information about nonheme iron deposition regardless of oxidation states in normal and pathological conditions. The perfusion-Turnbull + DAB method is specifically demonstrable of nonheme ferrous iron and the results from this method showed significant stores of nonheme ferrous iron in the hepatocytes, Kupffer cells, splenic macrophages, and gastric parietal cells of the rat. Since nonheme ferrous iron is considered to be critically involved in free radical generation, the perfusion-Turnbull + DAB method would visualize such populations of cells that are at risk from free radical damage.