Pseudocolor image processing of PEPCK subcellular distribution in rat hepatocytes shown with IGSS and epipolarization microscopy.

We read the report by Drs. Zimmer and Magnuson which appeared in the Journalof Histocbemutq and Cytochemistry 38:171,1990 (1). Their results are excellent, as shown in their photomicrographs, and indicate that there is a gradient ofphosphoenolpyruvate farb"ykioase (PEPCK) from periportal hepatocytes to the pericentral cells. It is known that several enzymes are involved in the regulation of gluconeogenesis (for the synthesis of both glucose and glycogen) and glycolysis (2). The finding of heterogeneous distribution of carbohydrate-metabolizing enzymes in the hepatocytes in different areas of the liver reflects variations in their functional state, and our observations (3,4) strongly support their study. However, in that study and others in the literature, we find that most light micrographs showing the immunohistochemical localization of the enzymes related to carbohydrate metabolism are at low or intermediate magnification using x 5-40 objective lenses and 5-10-pm thick sections. These conditions do not allow highresolution determination ofthe subcellular distribution of these enzymes. To reveal the subcellular distribution of PEPCK in individual hepatocytes at the light microscopic level, we have used goat anti-rat PEPCK serum as the priiary antibody, followed by immunogold localization with a gold-labeled secondary antibody, silver staining (IGSS), and high-resolution epipolarized light microscopy (5,6). Normal adult Sprague-Dawky rats were anesthetized with sodium pentobarbital and transcardially perfusion-fixed with cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, for 15 min. Slices (2 mm thick) of fixed liver were permeabilized in 1% Surfact Amps X-100 (Pierce; Rockford, IL) and 1% NH&I in half-strength PBS (0.1 M phosphate and 0.8% NaCI, pH 7.4) for 15 min at room temperature, then infiltrated with 30% sucrose in PBS overnight at 4°C. The cryoprotected slices were frozen on dry ice and cut into 10-pm cryosections that were thawed onto slides for immunocytochemistry. The immunogold-silver-stained sections were counterstained with 0.3% pyronine Y in water, dehydrated through an ascending ethanol series, and then embedded on the slides in a thin layer of plastic resin. After polymerization, the embedded sections were peeled off the slide, glued to a blank chuck, and cut into semi-thin sections (0.5 pm) with2 sapphire knife on an ultramicrotome. The resulting semi-thin sections were collected on glass slides and cmrslip-mounted with permount for epipolarization microscopy. For achieving the highest magnification we employed a x 100 objective lens and x 2 intermediate setting in a Nikon microphot FX microscope equipped with IGS filter ( x 1.25). The illumination is a combination ofa low intensity oftransmitted light with epipolarized light. The advantage of using both epipolarization illumination and normal light illumination is the ability to show the antigenic sites and the tissue prof& simultaneously. The PEPCK molecules were subcellularly localized by silver-enhanced gold particles. Under these conditions, the PEPCK antigenic site within the hepatocytes is highlighted by depolarized light (generated by gold-silver grains under the illumination of epipolarized light) and the cellular structure is shown by low-intensity