A Simple and Sensitive Histochemical Method for Calcium

Abstract

A technic is descr. for the histochemical definition of Ca. Thin tissue specimens not exceeding 2-3 mm. in thickness are fixed in 4 changes of 95% ethyl alcohol for 36 hrs., directly embedded into paraffin, and mounted on slides without adhesive. No exposure to dist. water is permitted during this stage. Mounted sections are then taken through 2 xylols, 2 absolute, and 2 95% ethyl alcohols and thence directly into 1% alizarin red S in 0.1% NH4OH, final pH 6.3-6.5, for 2 min. Excess stain is removed by gentle irrigation with distilled H2O (pH 5.8) then dehydrated with 2 95% and 2 absolute alcohols, clarified in 2 xylols and mounted in cedarwood oil. Ca deposits are shown as intensely-staining reddish-orange areas. Mg, and Fe when present as hemosiderin, will not be stained. The method is sensitive to Ca at a surface density of not more than 0.0004 ug./mm2. Estimates of the amts. of Ca present in tissues by this technic correlated well with the results obtained by microchemical quantitative estimates.