Three-dimensional and histochemical studies of peroxisomes in cultured hepatocytes by quick-freezing and deep-etching method

Abstract

Primary cultured mouse hepatocytes were treated with clofibric acid to induce peroxisome proliferation. They were briefly fixed with paraformaldehyde and centrifuged to prepare pellets. The hepatocytes were split open to remove cytoplasmic soluble proteins and fixed with glutaraldehyde. They were routinely incubated for catalase enzyme cytochemistry and fixed in osmium tetroxide. The specimens were quickly frozen, deeply etched and rotary shadowed by platinum and carbon. Peroxisomes were identified as osmium reaction products on replica membranes. In treated hepatocytes, the number of peroxisomes was considerably increased and smooth membranous structures resembling sER (referred to as 'peroxisome-forming sheets') showed hypertrophy. The rER associated with intermediate filaments was significantly decreased and 'peroxisome-forming sheets' appeared, being accompanied with budding and fragmentation of peroxisomes.