Histochemical investigations on the nature of large blood vessel endothelial and medial argyrophilic lines and on the mechanism of silver staining

Summary A study of the mechanisms involved in silver staining of blood vessels has been performed on the rabbit and rat aorta and vena cava, both in fixed and unfixed states. Pretreatment with cationic detergents, organic solvents, and solutions containing free iodide ions inhibited the silver staining. Anionic or neutral detergents, oxidizing agents, binders of such ions as Ca⁺⁺, Mg⁺⁺ and SO ₄ ^- failed to inhibit the staining. Staining of the intercellular gaps between endothelial cells and between smooth muscle cells could also be obtained if vessels were treated with a cationic detergent and bromocresol green, or by a modified Hale's colloidal iron technique. Silver lines could be returned to dechlorinated vessels, if treated with sodium chloride before silver nitrate staining, but not vice versa; by an extended treatment with dilute silver nitrate or with gold chloride following normal silver nitrate staining; and by treatment with heparin prior to silver staining. Dark chamber experiments have demonstrated that a photographic developer can take the place of light in the silver staining procedure and that a photographic fixer has the same effect on vessel silver staining as dechlorination.The obtained results have led to the hypothesis that silver staining of vessels occurs in two stages. In the first silver ions from silver nitrate are bound by polyanions located primarily in the intercellular gaps, and then reduced. This produces a network of reduced silver grains which, however, are still too sparsely aggregated to be visualized. Chloride ions in the tissues also bind and precipitate silver ions preventing their removal in subsequent rinsing procedures. In the second stage light (or a photographic developer) reduces the silver ions in silver chloride, producing a visible accumulation of metallic silver, but only around the silver grains reduced during the first stage, analogous to the photographic process.The possible existence and function of an intercellular cement substance is discussed in light of the evidence for the presence of polyanionic groups in the intercellular gaps.