ADVANTAGES OF DIGITONIN EXTRACTION TO REVEAL THE INTRACELLULAR STRUCTURE OF RAT GLOMERULAR PODOCYTES FOR HIGH-RESOLUTION SCANNING ELECTRON-MICROSCOPY

Kidneys of anesthetized rats were perfused with digitonin to extract c ytosolic proteins of glomerular podocytes so that the remaining intrac ellular structures could be examined by three-dimensional stereo high- resolution scanning electron microscopy (HRSEM). Cytoskeleton, consist ing of microtubules and intermediate filaments, was preserved with eac h applied concentration of digitonin. High concentrations of digitonin (1.0 mg/ml) produced a corrugated appearance in plasma membranes like ly 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 concent rations of digitonin (0.1 and 0.2 mg/ml) were less disruptive to both the plasma membrane and the Golgi complex. Mitochondria, rough endopla smic reticulum, coated vesicles, nuclear membrane, and chromatin were well preserved. Extraction with digitonin, at the optimal concentratio n and perfusion time, simultaneously maintains both the cytoskeleton a nd membranous organelles inside the cell and provides a method to eluc idate the interactions between these two components. Furthermore, digi tonin extraction should preserve antigenic sites, thereby allowing the localization of intracellular proteins by backscattered electron imag ing of immunogold labels in the scanning electron microscope. (C) 1993 Wiley-Liss, Inc.