Abstract
A simple pocedure for localizing calcium ions in tissues and in cells with glyoxal bis (2-hydroxy-anil) (GBHA) has been described. The GBHA molecule, in alcoholic solution made alkaline with NaOH, chelated with Ca, Ba, Sr, Cd, Cu, Co, and Ni ions, forming colored precipitates. All the colored GBHA complexes, with the exception of the red Ca-GBHA granules, were decolorized when immersed in a solution containing carbonate and cyanide. Of the methods available for demonstrating calcium ions, the sensitivity of the GBHA method was comparable only to the Scott-Packer electron microscope method; the latter, however, localized calcium plus magnesium while the GBHA method was specific for calcium ions.
In tissues that were fixed in 10% neutral formalin, alcohol-formalin or absolute alcohol, false localization due to binding of extraneous or diffused calcium ions to polyanionic sites was evident. The false localization of calcium ions was prevented by processing the tissues by the freeze-dry or the freeze-substitution method, dehydrating the tissue below the cutectic point of calcium chloride. The GBHA method was not able to demonstrate calcium ions present as insoluble salts, such as calcium carbonate or as apatite crystals, giving false negative results.