The Applicability of a New Schiff Base, Glyoxal Bis (2-Hydroxyanil),for the Cytochemical Localization of Ionic Calcium

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.

The entry of labelled calcium into the innervated region of the mouse diaphragm muscle

“…3A) show that the calcium was accumulated inside muscle fibres at the postjunctional region. It should be noted in connexion with this point that glyoxal bis(2-hydroxyanil) will stain only free calcium ions (Kashiwa & Atkinson, 1963), so the pattern of staining shown by PI. 3A reflects the distribution of free calcium ions which may be only a small fraction of the total calcium which is bound within the muscle cytoplasm.…”

“…It was necessary to photograph the slides soon after their preparation because the calcium stain faded overnight. Treatment of calcium stained sections with alcoholic Na2CO3/KCN solution, in order to remove the stain from divalent ions other than calcium (Kashiwa & Atkinson, 1963), made no difference to the appearance of the sections so this stage was omitted from the routine staining procedure.…”

“…0 4 % glyoxal bis (2-hydroxyanil) dissolved in methanol with 7-2 ml. 5 % NaOH (Kashiwa & Atkinson, 1963). Slides bearing the sections to be stained were dipped into this solution and placed in air so that the sections were covered by a thin film of staining solution.…”

Visual Detection of Calcium by GBHA Staining in Bitter Pit-affected Apples

“…GBHA is a highly specific Ca 2+ reagent widely used to determine calcium concentrations in water, milk and soil (Merck, 2003a,b,c). It was first used for the cytochemical identification of calcium by Kashiwa and Atkinson (1963) and later evaluated for calcium staining in pancreatic islets (Wolters et al, 1979).…”

Calpain and Cathepsins in the Skeletal Muscle of Inflammatory Myopathies

“…The cross-sections, 10 pm thick, were cut using a cryostat. For histochem ical analysis, serial frozen sections were stained with routine histo chemical methods [10], The C a;+ was stained with the glyoxal bis-(0-hydroxy-anil) methods [11], For the immunohistochemical study, serial sections were incu bated for 10 min in phosphate-buffered saline containing 10% nonfat dry milk, and washed. The specimens were then incubated in poly clonal or monoclonal antibodies for 1 h each at room temperature.…”