Printed in U.S.A. DIFFUSION ARTIFACTS IN CYTOCHEMISTRY OF CATALASE’

The various causes of diffusion artifacts in cytochemical localization of catalase in rat hepatic peroxisomes have been investigated. It is noted that storage of tissue in buffer after the fixation with glutaraldehyde is mainly responsible for diffusion of catalase from peroxisomes. In the vicinity of such peroxisomes with diffusion, the product of oxidation of 3, 3’-diaminobenzidine tetrahydrochloride (DAB) is localized on ribosomes and mem-branes of mitochondria and endoplasmic reticulum. In contrast, when the same tissue is reacted for catalase immediately after the fixation, no evidence of diffusion is observed. Prolonged incubation (up to 4 hr at 37#{176}C) and storage of tissue after the reaction with DAB (up to 9 months) do not cause any diffusion of the reaction product. Similarly, there is no evidence of diffusion around the peroxisomes when the complete incubation medium is perfused into the liver immediately after the fixation with glutaraldehyde (perfusion-incubation technique). These findings indicate that the diffusion of heme protein catalase, rather than the diffusion of oxidation product of DAB, is respon-sible for focal cytoplasmic staining, which is seen occasionally in the vicinity of some peroxisomes. The 3, 3’-diaminobenzidine tetrahydrochlo-ride (DAB) technique introduced originally by Graham and Karnovsky (18) for cytochemical localization of horseradish peroxidase has found wide application in diverse fields of cytologi