Nonheme-iron histochemistry for fight and electron microscopy: a historical, theoretical and technical review

We reviewed the methods of nonheme-iron histochemistry with special focus on the underlying chemical principles. The term nonheme-iron includes heterogeneous species of iron complexes where iron is more loosely bound to low-molecular weight organic bases and proteins than that of heme (iron-protoporphyrin complex). Nonheme-iron is liberated in dilute acid solutions and available for conventional histochemistry by the PerIs and Turnbull and other methods using iron chelators, which depend on the production of insoluble iron compounds. Treatment with strong oxidative agents is required for the liberation of heme-iron, which therefore is not stained by conventional histochemistr. The PerIs method most commonly used in laboratory investigations largely stains ferric iron, but stains some ferrous iron as well, while the Turnbull method is specific for the latter. Although the Turnbull method performed on sections fails in staining ferrous iron or stains only such parts of the tissue where iron is heavily accumulated, an in vivo perfusion-Turnbull method demonstrated the ubiquitous distribution of ferrous iron, particularly in lysosomes. The PerIs or Turnbull reaction is enhanced by DAB/silver/gold methods for electron microscopy. The iron sulfide method and the staining of redox-active iron with H2O2 and DAB are also applicable for electron microscopy. Although the above histochemical methods have advantages for visualizing iron by conventional light and electron microscopy, the quantitative estimation of iron is not easy. Recent methods depending on the quenching of fluorescent divalent metal indicators by Fe2+ and dequenching by divalent metal chelators have enabled the quantitative estimation of chelatable Fe2+ in isolated viable cells.