Use of Alizarin Red S for Histochemical Staining of Ca2+ in the Mouse; Some Parameters of the Chemical Reaction in vitro

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Abstract
Alizarin red S (ARS) is used for histological characterization of calcium. In order to improve the conditions of the use of the dye on biological materials, the chemical reaction between ARS and Ca2+ is studied in vitro. In aqueous solution ARS and Ca2+ ions precipitate to form brick-red deposits. For precipitation to occur a neutral pH (4 < pH < 8) is required; the stoichiometric ratio is 1:1. ARS reacts with calcium via its sulfonate and hydroxyl groups. The apparent solubility product is around 10∼7 Na+ and K+ ions do not interfere with equilibrium of the precipitation reaction but Mg2+ and soluble proteins such as bovine serum albumin react with ARS and form soluble complexes which can lead to a decrease in the ARS quantity available to react with the calcium ions.