STUDIES ON HISTOCHEMICAL ACYLATION PROCEDURES. I. PHENOLS

A series of tests of various acylation mixtures was made to determine their effectiveness in preventing protein and enterochromaffin azo coupling reactions at pH 8, usually with fast black K, C.I. No. 37190, (4-(p-nitrophenylazo)2, 5-dimethoxyaniline diazonium double zinc chloride). Reagents tested included acetyl chloride in ethyl acetate, p-toluenesulfonyl chloride in 50:50 acetone +5% aqueous borax, benzoyl chloride in pyridine and acetic anhydride in pyridine, ethanol, isopropanol and glacial acetic acid, with and without addition of perchloric and sulfuric acid as catalysts and dimethylaniline as a base. Acylations were regularly done at 23-25#{176}C, since it appears that temperature elevation tends to accelerate redissociation. Acylation times varied from 30 minutes to 32 hours and multiple intervals were employed in all comparative experiments. Histochemical material used comprised monkey duodenum for enterochromaffin and protein, rat stomach for soft keratin and protein, and guinea pig skin for hair, trichohyalin, keratohyalin and striated muscle. The most effective mixture employed was an equal volume mixture of absolute ethanol and acetic anhydride. This gave complete blockade of the azo coupling reaction at intervals of 1, 2, 4, 8 and 16 hours and almost complete in 30 minutes, both for enterochromaffin and for