HISTOCHEMICAL ACYLATION OF HYDROXYL AND AMINO GROUPS. EFFECT ON THE PERIODIC ACID SCHIFF REACTION, ANIONIC AND CATIONIC DYE AND VAN GIESON COLLAGEN STAINS

Acetylation of glycols in )yridine acetic anhydride mixtures is slow but apparently progressive. It may require 24-48 hours for comnplete blockade of the periodic acid Schiff reaction of some objects. It is somewhat, but not markedly, accelerated by addition of 0.4-0.5% of concentrated sulfuric acid by volume. The blockade is PrOml)tlY and fairly completely reversed by sal)Onification in 1% KOH/70% alcohol (20-30 mm) and in a 20% dilution of 28% amnionia with absolute alcohol (16-24 hours). Partial unblocking also occurs in 0.1 M sulfuric acid/methanol (2 hours, 60#{176}C). Acetylation of glycols occurs also in 25-50% solutions in absolute ethanol. Here maximal blocking effect is observed at 4-8 hours at 25#{176}C, with apparent rehvdrolysis thereafter. The maxinsuns effectiveness is similar to that of 16-24 hour acetylation in pyridimse. Additioms of dinsethylaniline as a base to take up liberated acid is without appreciable effect. Addition of sulfuric acid (0.5% v/v) is deleterious an(! inclusioms of KOH in the acetylatioms bath results its concurrent saponification and failure of the reaction. Acetvlation in 10% acetyl chloride in ethyl acetate is about as effective as that in 25% acetic anhydride/pyridine. Tosylation in 2.5% p-toluenesulfonyl chloride in equal volumsses of acetone and 5% borax was less effective than the l)yridine acetic anhydride reagents in blocking the periodic acid Schiff reaction. \‘erv rapid acylation and complete blockade of the PeriO(Iic acid Schiff reaction are attained in 25% acetic anhydride glacial acetic acid mixtures contaimsing small amounts of concentrated sulfuric acid. With 0.25% v/v (46 mM) sulfuric acidl as little as 3 minutes is ade-