Rat livers perfused with 0.5% Triton X-100 for 15 to 90 min followed by 1% sodium dodecyl sulfate for 30 min were studied by electron microscopy and polyacrylamide gel electrophoresis. After 15 min of perfusion, a rich network of intermediate filaments and microtubules was visualized in the cytoplasm of hepatocytes. Using stereopairs, branching was visualized. Connections of filaments were noted with nuclei, centrioles, microtubules, vesicles, and rough endoplasmic reticulum. The existence of connections supported the concept that intermediate filaments may function to integrate mechanically the cytoplasmic space as postulated by Lazarides.In 1975, we identified filaments in hepatocytes, which did not decorate with heavy meromyosin (1,2). Phillips et al. (3) described similar filaments in isolated preparations of bile canalicuii. We referred to these filaments as intermediate filaments according to the nomenclature of Ishikawa et al. (4). These filaments were further characterized using digitonin extraction (5) of isolated hepatocytes and detergent extraction of glutaraldehyde-fixed preparations (6). Jahn has recently visualized this filament system in electron microscopic hepatocytes using negatively stained frozen sections of liver (7) and liver cells extracted with detergent (8). Denk et al. (9) visualized an elaborate system of intermediate filament antigen in hepatocytes using the immunofluorescent antibody technique. However, despite these elegant studies, the intermediate filament cytoskeleton has not been easily visible by transmission electron microscopy when conventional fixation and staining of hepatocytes is performed.We have attempted to design a method to visualize the hepatocyte cytoskeleton by transmission electron microscopy. Attempts were made using nuclear isolates, broken cell preparations, glycerinated liver, and detergent-extracted isolated hepatocytes or minced liver, but the results were not optimal. Finally, we were successful when we perfused intact liver with a detergent solution. The results of the perfusion method are the subject of this report.
MATERIALS AND METHODSSprague-Dawley male rats of 350 to 400 gm were used for the perfusion studies. Prior to perfusion, the rats were maintained on Purina Rat Chow ad libitum.After preliminary experiments were completed to determine the best time sequence for sampling the liver during perfusion, three rats were utilized for liver perfusion-extraction experiments as follows.The rats were anesthesized with pentobarbital (5 mg per 100 gm body weight, i.p.) using a 5% solution. At iaparotomy, a liver biopsy was taken for formalin fixation, paraffin embedding, and hematoxylin-eosin staining for light microscopy. A portion of the biopsy was taken for electron microscopy using immersion fixation and en bloc staining using 2% uranyl acetate in 10% ethanol. Tissues were dehydrated in ethanols, embedded in Spurr's resin, and viewed in a Zeiss 109R and Phillips 400 electron microscope. Stereopairs were made using a goniometer stage on thick sections (40...
