The Effect of Decalcifying Fluids on the Staining of Epithelial Mucins by Alcian Blue

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Abstract
Eleven decalcifying fluids were tested for rate of decalcification and for their effect on subsequent staining by each of 4 variations of the Alcian blue technique. Tests for decalcification were made on 5 mm slices of fixed adult rat heads. Tests on staining were made on 5 μm sections cut after celloidin-paraffin embedding of the slices. Evaluation of staining was based chiefly on the reaction of acid glycoproteins in goblet cells in epithelium and in mucous glands of the upper respiratory tract. A 15% aqueous solution of ethylenediaminetetraacetic acid, sodium salt at pH 7.0 (Versenate), acting for 3 days at 36 C gave the best results as judged by either speed of decalcification or by quality of Alcian blue staining or that given by haematoxylin and eosin. Uniformly good results were also given by Kristensen's equal-parts mixture of 8 N formic acid and 1 N sodium formate and by 10% formic acid—either at 20–25C. Versenate has the additional advantage that tissue can be left in it over long periods.