Pyrogallol red-vanadium complex—a new stain for electron microscopy

Abstract

We report on the application of a pyrogallol red-vanadium complex (PR-V) for ultracytochemical staining of proteinaceous structures in animal tissues and cell cultures. This dye may be used as a general purpose stain in electron microscopy. In contrast to osmium tetroxide, the price of the material is low and no toxic vapors are produced. The PR-V complex was prepared by addition of vanadium (IV) oxide sulfate to pyrogallol red dissolved in acetate buffer (pH 5.6). The formation of the complex was indicated by a color change from purplered (λ_max=520 nm) to violet (λ_max=539 nm) which occurred at equimolar concentrations of the dye and the metal salt. Under these conditions PR-V was stable for several days. The mechanism of PR-V binding was checked in dot blots using different proteins as well as heparin for control. While heparin remained unstained, proteins were stained in a dose-dependent manner. Deamination of proteins with nitric oxide strongly reduced PR-V staining in dot blots as well as in cell cultures. Optimal staining results of animal cells and tissues were obtained in specimens that had been mildly fixed for at least 1 h or longer with a mixture of 0.1% glutaraldehyde and 1.0% paraformaldehyde dissolved in phosphate-buffered saline, pH 7.2, washed with acetate buffer, pH 5.6, and subsequently treated with PR-V in the presence of 50% ethanol at room temperature. Control specimens without PR-V but treated en bloc with uranyl acetate or sodium molybdate showed similar contrast but less details in the ultrastructure of the tissue. All specimens were embedded in epoxy resin and ultrathin sections were stained conventionally with uranyl and lead salt solutions. In electron micrographs, membrane-associated particles, stress fibers and filaments of the cell cortex, collagen fibrils, tight junctions and desmosomes, and other proteinaceous components were clearly visualized only in the PR-V-treated speciments. In conclusion, the ability to bind selectively and specifically to proteinaceous structures makes PR-V a versatile stain to study the localization and distribution of these structures in cells and tissues at the ultrastructural level.