IMMUNOGOLD-SILVER STAINING AND EPIPOLARIZED LIGHT-MICROSCOPIC DETECTION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE AND GLYCOGEN-PHOSPHORYLASE IN RAT-LIVER

The subcellular distribution of enzymes related to carbohydrate metabo lism was determined in sections of paraformaldehyde fixed and polyethy lene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) seru m and rabbit anti-rat glycogen phosphorylase (GP) serum were used as p rimary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary ant ibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining usin g appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunore active molecules were found only in glycogen-containing areas of the c ytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the prima ry antibody in the procedures. Visio-Bond semithin (0.35-1.0 mum) sect ions provided higher resolution for subcellular immunostaining of PEPC K and GP than cryosections of 10 mum. Epipolarized light microscopy pr ovided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization o f the tissue morphology.