Differentiation of Nucleic Acids by Staining at Controlled pH and by A Schiff-Methylene Blue Sequence

Abstract

Fixation with Bouin''s fluid preserves cytoplasmic and nucleolar ribonucleic acid (RNA) particularly well. RNA may be demonstrated preferentially in Bouin fixed tissue by staining with 0.02% thiazine dye in aqueous McIlvaine phosphate-citrate buffer between pH 3 and 4. Methylation blockage of basophilia other than that of nucleic acids permits staining of RNA with thiazine dyes near neutrality. The deoxyribonucleic acid (DNA) of chromatin undergoes a Feulgen type hydrolysis in the tissue block during 24 hr fixation with Bouin''s fluid. This hydrolysis by picric acid permits Schiff staining of the DNA without further acid hydrolysis. Consequently after Bouin fixation it is possible to demonstrate DNA and RNA specifically by a Schiff-methylene blue sequence. Thus a Schiff stain without further acid hydrolysis followed by 0.02% methylene blue in phosphate-citrate buffer at pH 3.0 to 3.5 colors DNA magenta in contrast to the blue of RNA.