CYTOCHEMICAL APPLICATION OF TRIS(2,2'-BIPYRIDINE) RUTHENIUM(II) - FLUORESCENCE REACTION WITH SULFATED POLYANIONS OF MAST-CELL GRANULES

We describe the use of tris (2,2'-bipyridine) ruthenium (II) (Rubipy) as a cationic fluorochrome for cytochemical and histochemical studies. After staining with Rubipy, mast cell granules (MCGs) and lymphocyte nuclei (LN) from mouse peritoneal cavity and human breast carcinoma sh owed intense orange fluorescence and no fading under blue or blue-viol et exciting light. Staining at low pH (<2) or pre-treatment with Al3ions strongly diminished the fluorescence of LN, whereas that of MCG w as less affected. Ca2+ and Ba2+ ions only diminished MCG fluorescence. Blots of DNA, pectic acid, heparin, and other sulfated polysaccharide s stained with Rubipy showed high emission, which was reduced in DNA a nd pectic acid staining at low pH. Studies with chemically modified he parins suggested that O-sulfates were more important than N-sulfates i n Rubipy-heparin interactions. These results are in agreement with an ionic binding mode between Rubipy and heparin. A very suitable method for mast cell detection was found with Mayer's hematoxylin before Rubi py staining, which could be of great value for histopathological studi es. This procedure allowed visualization of the mast cells by fluoresc ence microscopy, and nuclei and tissue morphology were easily visualiz ed under bright-field illumination.