title: Advantages of digitonin extraction to reveal the intracellular structure of rat glomerular podocytes for high-resolution scanning electron microscopy.
abstract: Kidneys of anesthetized rats were perfused with digitonin to extract cytosolic proteins of glomerular podocytes so that the remaining intracellular structures could be examined by three-dimensional stereo high-resolution scanning electron microscopy (HRSEM). Cytoskeleton, consisting of microtubules and intermediate filaments, was preserved with each applied concentration of digitonin. High concentrations of digitonin (1.0 mg/ml) produced a corrugated appearance in plasma membranes likely due to the formation of digitonin-cholesterol complexes. At 1.0 mg/ml digitonin, the Golgi complex became vesicularized, and mitochondria were well extracted and their ultrastructure preserved. Lower concentrations of digitonin (0.1 and 0.2 mg/ml) were less disruptive to both the plasma membrane and the Golgi complex. Mitochondria, rough endoplasmic reticulum, coated vesicles, nuclear membrane, and chromatin were well preserved. Extraction with digitonin, at the optimal concentration and perfusion time, simultaneously maintains both the cytoskeleton and membranous organelles inside the cell and provides a method to elucidate the interactions between these two components. Furthermore, digitonin extraction should preserve antigenic sites, thereby allowing the localization of intracellular proteins by backscattered electron imaging of immunogold labels in the scanning electron microscope.
authors.affiliation: Canadian Genetic Diseases Network, Faculty of Medicine, University of Toronto, Ontario.
authors.forename: R J
authors.initials: RJ
authors.lastname: Temkin
authors.forename: D Y
authors.initials: DY
authors.lastname: So
authors.forename: P J
authors.initials: PJ
authors.lastname: Lea
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure; D003599:Cytoskeleton / Q000648:ultrastructure; D004072:Digitonin; D007382:Intermediate Filaments / Q000648:ultrastructure; D007678:Kidney Glomerulus / Q000166:cytology / Q000648:ultrastructure*; D008297:Male; D008855:Microscopy, Electron, Scanning; D008870:Microtubules / Q000648:ultrastructure; D015388:Organelles / Q000648:ultrastructure; D010477:Perfusion; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D004072:Digitonin