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 Perls 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 histochemistry. The Perls 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 Perls 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 H(2)O(2) 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 Fe(2+) and dequenching by divalent metal chelators have enabled the quantitative estimation of chelatable Fe(2+) in isolated viable cells.
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.
Visually responsive neurones of the cat's suprageniculate nucleus were categorized according to their responses to a variety of different types of light stimuli. Their velocity preferences were assessed quantitatively and have been found to predominate in the high range. The positions and dimensions of their receptive fields were determined and these tended to be found within a zone between the vertical meridian and 30-35 degrees. Receptive fields had mean diameters smaller than those reported by others for the pulvinar-LP complex. From these and other electrophysiological data, inferences have been made of the likely sources of afferents providing visual driving. A proposal is offered for SGn visuotopic organization based on correlations of receptive field location with cell position, as determined through electrode track reconstructions.
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