CYTOCHEMICAL LOCALIZATION OF NUCLEIC ACIDS BY AN ACRIFLAVINE-PHOSPHOTUNGSTATE COMPLEX FOR FLUORESCENCE MICROSCOPY AND ELECTRON MICROSCOPY

Abstract

An acriflavine-phosphotungstate complex (A-PTA) was synthesized from a reaction between acriflavine and phosphotungstic acid, with the former in excess. The purified product is fluorescent and electron-dense, and was studied for its use as a cytochemical reagent for fluorescence microscopy and electron microscopy. A selectivity for nucleic acids and sulfated cerebroside esters that excludes sulfated glycoaminoglycans is proposed. The reaction for fluorescence microscopy is rapid, sensitive and reproducible. The electron-dense A-PTA complex localized nucleic acids for electron microscopy. Known sites of DNA and RNA in cells of pancreatic, cerebellar and testicular tissue have been demonstrated with the reaction. The reaction is administered as a single step, interposed in the electron microscopic preparative procedure, and can be used in either an alcoholic or an aqueous system. The alcoholic A-PTA reaction produced adequate contrast on short duration (2 hr); the aqueous block reaction was found most satisfactory when prolonged to 48 hr. Uranyl acetate, phosphomolybdic acid and phosphotungstic acid can be used as supplementary stains in addition to the A-PTA reaction. Under these conditions, DNP and RNP sites were demonstrated. The A-PTA complex is a prototype of the coupled dye-metal histochemical reagents, and its selectivity for nucleic acids represents a new approach in cytochemistry. Its reactions can be investigated by fluorescence microscopy and electron microscopy, and in solution. Other dye-metal reagents can probably be formulated on a similar design as detection systems for lipids, polysaccharide or specific groups in proteins.