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 challenge to the morphologist. In the course of studies of the distribution of the glucose transporter GLUT4, in muscle, we have compared different preparative procedures, for both light (LM) and electron microscopy (EM) immunocytochemistry. 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 establish fixation and permeabilization conditions for EM immunogold labeling of the fibers. We find that a simple fixation with depolymerized paraformaldehyde alone, followed by permeabilization with 0.01% saponin, offers the best compromise between the conflicting demands of unhindered tissue penetration and morphology preservation.
Ralston
E
E
Laboratory of Neurobiology, NINDS, National Institutes of Health, Bethesda, MD 20892-4062, USA. esr@codon.nih.gov
Ploug
T
T
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure; D005557:Formaldehyde; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4; D016591:Histocytological Preparation Techniques; D007150:Immunohistochemistry; D008297:Male; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D009004:Monosaccharide Transport Proteins / Q000032:analysis*; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000648:ultrastructure*; D009124:Muscle Proteins; D018482:Muscle, Skeletal / Q000648:ultrastructure; D009469:Neuromuscular Junction / Q000648:ultrastructure; D051381:Rats; D017208:Rats, Wistar; D013194:Staining and Labeling
D051275:Glucose Transporter Type 4; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D005557:Formaldehyde; D005947:Glucose