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 metabolism was determined in sections of paraformaldehyde fixed and polyethylene glycol-1540-embedded rat liver and in cryostat sections. For this purpose, goat anti-rat phosphoenolpyruvate carboxykinase (PEPCK) serum and rabbit anti-rat glycogen phosphorylase (GP) serum were used as primary antibodies to localize the corresponding antigens. The primary antibodies were localized by 5 nm colloidal gold labeled secondary antibodies (either rabbit anti-goat IgG for PEPCK or goat anti-rabbit IgG for GP), and the gold particles were enhanced by silver staining using appropriate development reagents. The silver enhanced gold particles were detected by epipolarized light microscopy. PEPCK and GP immunoreactive molecules were found only in glycogen-containing areas of the cytosome of hepatocytes, and not in other cells. No immunocytochemical staining of hepatocytes was found when normal serum replaced the primary antibody in the procedures. Visio-Bond semithin (0.35-1.0 micron) sections provided higher resolution for subcellular immunostaining of PEPCK and GP than cryosections of 10 microns. Epipolarized light microscopy provided detection at high sensitivity of the gold-labeled antibody, and combined with transmitted light, allowed simultaneous visualization of the tissue morphology.
Gao
K
K
Department of Anatomy and Cell Biology, University of Cincinnati College of Medicine, OH 45267.
Morris
R E
RE
Giffin
B F
BF
Cardell
R R
RR
D000818:Animals; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D008297:Male; D008859:Microscopy, Polarization; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000276:immunology / Q000378:metabolism*; D006005:Phosphorylases / Q000276:immunology / Q000378:metabolism*; D016613:Plastic Embedding; D051381:Rats; D017207:Rats, Sprague-Dawley; D013347:Subcellular Fractions / Q000201:enzymology
D006005:Phosphorylases; D010729:Phosphoenolpyruvate Carboxykinase (GTP)