Exact ultrastructural localization of glutathione peroxidase in normal rat hepatocytes: advantages of microwave fixation.

Glutathione peroxidase (GSH-PO), a highly soluble, selenium-dependent enzyme metabolizing lipid peroxides, is allegedly distributed in both the cytosol and mitochondria. With the pre-embedding method of immunoelectron microscopy for GSH-PO employing conventional immersion-fixation, the nuclei of rat hepatocytes stain positively, whereas mitochondria are negative. Such observations are inconsistent with the results of biochemical and immunoblot analyses using isolated subcellular fractions. In the present study, we employed the combination of microwave irradiation and fixation in 4% paraformaldehyde (PFA), with or without 0.1% glutaraldehyde (GA), to enhance the accuracy of ultrastructural localization of GSH-PO in rat liver. A small block of liver was irradiated by microwave for 10 sec in cold cacodylate-buffered 4% PFA containing 0.1% GA. After further immersion of the tissue in 4% PFA at 4 degrees C for 1-6 hr, the standard procedure for pre-embedding immunoelectron microscopy was employed. We observed partial inhibition of artifactual diffusion of cytosolic GSH-PO into the nuclei and consistent GSH-PO localization in mitochondria. Dual localization of this enzyme in the cytosol and mitochondria of normal rat hepatocytes was thus confirmed.