Kx. Gao et al., IMMUNOGOLD-SILVER STAINING AND EPIPOLARIZED LIGHT-MICROSCOPIC DETECTION OF PHOSPHOENOLPYRUVATE CARBOXYKINASE AND GLYCOGEN-PHOSPHORYLASE IN RAT-LIVER, Histochemistry, 99(5), 1993, pp. 341-346
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.