Immunogold electron microscopic localization of phosphoenolpyruvate carboxykinase in rat liver

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-limiting gluconeogenic enzyme which catalyzes the conversion of oxaloacetate to phosphoenolpyruvate. A gradient of PEPCK from periportal hepatocytes to pericentral hepatocytes has been demonstrated in normal rat liver; however, the subcellular distribution of PEPCK molecules within hepatocytes could not be determined from these light microscope studies.We have used two immunogold histochemical approaches to study the subcellar distribution of PEPCK in normal rat hepatocytes. In the first procedure 10μm cyrosections of 4% paraformaldehyde perfusion-fixed rat liver were collected on silanated slides and immunostained with goat anti-rat PEPCK (normal goat serum served as a control), and incubated in a secondary antibody (5nm gold-labeled rabbit anti-goat IgG) and a tertiary antibody (5nm gold-labeled goat anti-rabbit IgG). The immunogold stained samples, with or without silver enhancement (IGSS), were counterstained with pyronine Y, re-embedded in Visio- Bond and removed from the slides and semithin sectioned for epipolarized illumination combined with transmitted light microscopy (Figs. 1 and 2), or re-embedded in epoxy resin and sectioned for electron microscopy (EM, Fig. 4) In the second procedure 25, 50 and 100 μm free-floating vibratome sections of well fixed rat liver (4% paraformaldehyde plus 0.5% glutaraldehydeperfusion-fixed, immersion fixed overnight at 4°C) were immunogold stained using a 5nm gold labeled secondary antibody, with and without IGSS, osmicated, embedded in epoxy resin and sectioned for EM (Figure 3).