ACETYLATION AND NITROSATION OF TISSUE AMINES IN HISTOCHEMISTRY

Acetylation to prevent the reaction of 1 ,2 glycols to the periodic acid Schiff procedure was introduced into histochemistry by McManus and Cason (20) in 1950. Seligman (25) commented that amines would also be acetylated, which MacManus admitted, but stated that the saponification by potassium hydroxide restored the reactivity of hydroxyls but would not decompose acetylated arnines. This statement has been widely accepted and various authors have used it for exclusion of amines as the reactive groups (6, 9, 10). It has further been stated that amines are more easily acetylated than glycols and that undiluted acetic anhydride or its fresh aqueous solutions could be used for this purpose, whereas alkali or preferably pyridine solutions or hot acetic anhydride are required to acetylate hydroxyls. In the same year (1950) Monn#{233}and Slautterback (21) reported that the staining of the basic protein of the chromidia of the Bouin-flxed sea urchin egg with the azocarmine G of the MalloryHeidenhain “Azan” procedure was unaltered by nucleic acid extraction and digestion procedures. However, this acidophilia was completely destroyed by 1-12 hour exposure at room temperature to Van Slyke’s acetic acid, sodium nitrite reagent, or by 12 hours at room temperature in 5% chloramine T as deamination procedures. Further, the acidophilia was destroyed by acetylation for 3-12 hours at 80#{176} in undiluted acetic anhydride; 1-3 days at 4#{176}C. abolished the blue staining of yolk granules but not the red chromidial reaction. The acetylation required in this series for prevention of the periodic acid Schiff reaction of yolk was 12 hours at 80#{176}. In 1952 I reported (15) the shift from acidophilia to basophiia of erythrocytes on nitrous acid deamination of alcohol fixed blood films, using Giemsa stain, and noted that this shift did not occur in formaldehyde fixed tissue with the deamination conditions used on blood films. While various procedures introduced for demonstration of protein amino groups have used nitrous acid deamination or acetylation or both as control procedures, no systematic study appears to have been made utilizing the well-known nitrous acid reactions for the discrimination between primary, secondary and tertiary amine groups. Likewise, the acetylation-deacetylation procedure of McManus and Cason appears to have been applied only to Criegee cleavage reactions for glycols, using Schiff reagent to demonstrate the aldehydes, and not to methods which might with some probability demonstrate amine groups.