A Demineralization Procedure for Enzymatic Histochemical Use: A Quantitative Succinic Dehydrogenase Assay

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Abstract
Thinnest practical slices of bones or teeth are suspended at 4 C in a 10% solution of EDTA in 0.1 M tris buffer brought to pH 6. 95 with KOH pellets. The solution is stirred moderately and continuously with a magnetic stirrer until specimens are demineralized. Unwashed specimens, taken directly from the demineralizing fluid, are frozen on a block of CO2 ice, mounted on a tissue carrier and sectioned at 6 [mu] in a cryostat. Histochemical stains conducted on sections stored in a slide holder enclosed in a plastic bag in a refrigerator for as long as 2 wk were successful. Quantitative studies on the preservation of succinic dehydrogenase showed nearly all of it present in specimens demineralized for 2 days, and approximately 50% remaining in specimens demineralized for 7 days. Qualitative studies with other dehydrogenases indicate that several may be similarly affected.