HISTOCHEMICALLY SELECTIVE ACIDOPHILIA OF BASIC NUCLEOPROTEINS IN CHROMATIN AND NUCLEOLI AT ALKALINE pH

Chromatin and nucleoli of tissues fixed with formalin-free, mercurial or alcoholic solutions, have the unique property of staining metachromatically with the acid dye Biebrich scarlet in the restricted pH range 9.0-10.0. The nuclear structures lack affinity for fast green FCF under these conditions so that a mixture of Biebrich scarlet and fast green stains chromatin and nucleoli a characteristic orange-red at pH 9.5. A Feulgen-Biebrich scarlet sequence distinguishes magenta DNA in chromatin and orange-red basic protein in nucleoli. Although nuclei in tissues fixed with the buffered mercurial show strong affinity for the basic dye alcian blue, nuclei in formalin-fixed tissue do not. In formalin-fixed, trichloroacetic acid (TCA)-extracted tissues, chromatin stains with Biebrich scarlet or fast green but nucleoli do not. The acidophilia induced by TCA may in part depend on the fact that deoxyribonucleic acid (DNA) is removed by this treatment. However such tissues lose the acidophilia of the chromatin when treated again with formaldehyde but regain the acidophilia after re-extraction with TCA, indicating removal of formaldehyde condensates from amines by TCA. The affinity of chromatin and nucleoli for Biebrich scarlet at pH 9.5 is highly susceptible to destruction by relatively brief nitrosation, acetylation or exposure to formaldehyde. Extraction with a sulfuric acid-mercurial reagent also eliminates this staining; but other solvents used for isolation of histone do not, except that 1 N HCl has such an effect when employed on sections of tissue fixed with HgCl2. After mild treatment with TCA, exposure to pH 9.5 buffer extracts DNA whereas buffer at pH 6.0 has no such effect.