Pre-embedding staining of single muscle fibers for light and electron microscopy studies of subcellular organization

Skeletal muscle fibers are large, multinucleated cells which pose a challen ge to the morphologist. In the course of studies of the distribution of the glucose transporter GLUT4, in muscle, we have compared different preparati ve procedures, for both light (LM) and electron microscopy (EM) immunocytoc hemistry. Here we show that pre-embedding staining of single teased fibers, or of single enzymatically dissociated fibers, has several advantages over the use of sections for observing discrete patterns that extend over long distances in the cells. We report on an optimization study carried out to e stablish fixation and permeabilization conditions for EM immunogold labelin g of the fibers. We find that a simple fixation with depolymerized paraform aldehyde alone, followed by permeabilization with 0.01 % saponin, offers th e best compromise between the conflicting demands of unhindered tissue pene tration and morphology preservation.