Effect of Fixation with Reduced Osmium Tetroxide upon the Antigenicity of Liver Catalase and Erythrocyte Esterase D

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Effect of reduced OsO₄ fixation on the antigenicity of rat liver catalase and erythrocyte esterase D was investigated. After fixation with mixture of 4% paraformaldehyde and 0.2% glutaraldehyde, rat liver and erythrocytes were post-fixed with reduced osmium (0.1% to 1.0%) for 1 hr. Some erythrocytes were fixed with 0.5% reduced osmium for various times (10 min to 60min). The antigenicity of both enzymes was expressed as labeling density (gold particles/μm² of peroxisomes or erythrocytes). Fixation with reduced OsO₄ greatly affected the antigenicity of both enzymes. Approximately 80% of catalase antigenicity was lost at 0.4% reduced OsO₄, whereas about 80% of esterase D antigenicity was lost at 0.2%. Positive contrast of membrane structures clearly appeared at 0.6% OsO₄. About 80% loss of the esterase D antigenicity was observed at 10min after fixation with 0.5% reduced OsO₄. Afterwards, the antigenicity decreased very slowly. The data indicates that at the concentration of reduced OsO₄ which stains the membrane structures positive contrast, 80% of the antigenicity is lost in the cases of catalase and esterase D.