Hematoxylin substitutes. A study of phenocyanin TC and the use of afterchrome mordanting in histology.

A practical, simple synthesis of the obsolete mordant dye, phenocyanin,* was devised, proceding from gallocyanin and resorcinol with acid and heat. The dye gave promise of good performance in metachrome iron mixtures, but because of excessive precipitation, the practice of afterchroming was taken from textile dyeing usage, and proved very successful. Of a number of metallic salts tried, Fe II proved to be the best, then Cu II and Fe III. The stain acts as a cationic dye on nucleic acids and other acidic tissue components: acid mucins, cartilage, mast cells, corpora amylacea, etc. The afterchroming process renders the stain much more resistant to various extraction agents and even moderately resistant to acid alcohol. Color values are quite comparable to those obtained with hematoxylin–eosin when an eosin counterstain is used. Nuclei, basophilic cytoplasm, Nissl granules, and bacteria color dark blue; cartilage, mast cells, and some acid mucins, deep violet. Staining with the 1% solution was essentially unaltered from neutrality down to 1.2 N HCl, pH 0.68, and dilution of the dye to 0.05% in 1% conc. HCl (pH 1.2) still gave excellent nuclear, RNA, and mast-cell staining. At 0.02% and pH 1.2, nuclear staining was distinctly weakened; mast cell granules were still dark violet.