Improved staining of negative binding sites with ruthenium red on cryosections of frozen cells.

Hexavalent cationic dye ruthenium red (RR) binds to anionic sites of cellular components, predominantly to the surface coat rich in glycoconjugates, and can be used as a marker of negative binding sites. Due to limited penetration of RR only superficial layers of cells are stained satisfactorily. To improve RR staining of L1210 leukemic cells isolated from culture and concentrated by centrifugation, cryosections of frozen cells were treated by RR to expose simultaneously all the cells and their components to the dye treatment. Cells were fixed with 2% glutaraldehyde in cacodylate buffer (CB), soaked in 2.2 mol/l sucrose and frozen by plunging into liquid nitrogen. Ultrathin cryosections were cut at a temperature of -90 degrees C, transferred to Formvar coated copper grids, postfixed with 1% OsO4 and stained with 0.05% RR in CB for 60-120 min. After removing RR solution with filter the grids were dried and examined electron microscopically. The resulting staining was a combination of a negative contrast (the plasma membrane and membranes of intracellular organelles) and of a positive contrast (cytoplasmic matrix and the extracellular coat). RR staining of negative binding sites on cryosections has proved useful for uniform exposure of all cells and cellular compartments to the dye and especially of external coat containing glycoconjugates.