A Tribasic Stain for Thin Sections of Plastic-Embedded, OsO4-Fixed Tissues

Abstract

Thin (0.5–1 μ) sections of plastic-embedded, OsO₄-fixed tissues were attached to glass slides by heating to 70 C for 1 min. A saturated solution combining toluidine blue and malachite green was prepared in ethanol (8% of each dye) or water (4% of each dye). Methacrylate or epoxy sections were stained in the ethanol solution for 2–5 min. The water solution was more effective for some epoxy sections (10–80 min). Epoxy sections could be mordanted by 2% KMnO₄, in acetone (1 min) before use of the aqueous dye, reducing staining time to 5–10 min and improving contrast. Aqueous basic fuchsin (4%) was used as the counter-stain in all cases; staining time varied from 1–30 min depending upon the embedding medium and desired effects, methacrylate sections requiring the least time. In the completed stain, nuclei were blue to violet; erythrocytes and mitochondria, green; collagen and elastic tissue, magenta; and much and cartilage, bright cherry red. Sections were coated with an acrylic resin spray and examined or photographed with an oil-immersion lens.