Intracellular Binding of Ruthenium Red in Frog Skeletal Muscle

During the course of an electron microscope study of the staining of skeletal muscle cell surfaces by ruthenium red (RR), it was noticed that certain staining conditions brought about a reproducible pattern of intracellular staining. The purpose of this communication is to describe that staining pattern and to discuss its implications with regard to the location of highly anionic regions within muscle cells. The basic staining procedure was that described by Luft (1971 a). Bundles of 5 20 fibers dissected from frog semitendinosus muscles were exposed to RR (0.1 or 1.0 mg/ml) during a l-h period of fixation with 3.5% glutaraldehyde in 100 mM sodium cacodylate buffer, during a rinse in caco-dylate buffer containing 10% sucrose, and during a 2-h period of postfixation with 1% osmium tetrox-ide in 100 mM cacodylate buffer. Cross sections were examined under the light microscope to make sure there was a complete layer of RR staining around each cell, a requirement for reproducible intracellular staining. The critical parameter turned out to be the duration of exposure to RR in the rinse. Periods of time less than 1 h were not sufficient to produce intracellular staining, but when the muscles were left overnight in the rinse containing RR the complete pattern of intracellu-lar staining did appear. and was recrys-tallized once or twice by the method of Fletcher et al. (1961) and washed with ethanol and ether to bring the molar extinction coefficient at 5,300 A in 0.1 M ammonium acetate to about 40,000. The commercial material had extinction coefficients in the range of 7,000 17,000 when corrected for absorbance at 3,600 A and 7,340 A by the method of Luft (1971 a). The pure RR is reported to have an extinction coefficient of 68,000 (Fletcher et al., 1916; Luft, 1971 a). Although the purity of the RR FIGURE 1 Triad from muscle fixed in the conventional way with glutaraldehyde followed by osmium postfixation. Muscle was held overnight in the cacodylate rinse between fixation and postfixation, x 47,600. Scale 0.5 um. FIGURE 2 Triad from muscle exposed to RR (1 mg/ml) overnight in rinse between glutaraldehyde fixation and osmium postfixation. Fixation and postfixation solutions also contained RR. x 94,200. 0.3 #m. FIGURE 3 Triad from muscle exposed overnight to Ringer's containing RR (0.2 mg/ml) before glutaraldehyde fixation. Fixation was followed by a brief rinse and osmium postfixation, x 48,400. Scale 0.5 /.tm. FIGURE 4 Cross section of …