Mitochondrial Fixation for the Detection of Cytochrome Oxidase Activity Using Microwave Irradiation

We examined cell fixation with microwave irradiation (MWI) used in cytochemistry. MWI was applied to blocks of about 1mm3 of mouse parotid glands at 500W for about 5 sec in a fixative at 37°C. The activities of endogenous peroxidase and mitochondrial cytochrome oxidse were demonstrated by usinng the DAB method with 3,3'diaminobenzidine (DAB) and 0.01% H2O2. Under electron microscopy, peroxidase activity was localized in the nuclear envelope, endoplasmic reticulum and sectetory granules. However, mitochondria cytochrome oxidase activity seemed to be rather weak against the MWI at 37°C. Moreover, suspension of isolated hamster liver mitochondria was fixed by MWI and also demonstrated cytochrome oxidase activity by using the cytochemical methods with DAB, cytochrome c, catalase and sucrose. Such mitochondrial fractions were subjected to 6-second MWI given 10 or 18 times with an interval of 10 seconds with and without a chilled water bath. The final temperature of each fixative was kept at about 10°Cor rose to about 37 and 55°C. When we took care to keep the temperature below 10°C, the DAB reaction products accumulated in the mitochondrial intermembrane-intracristal space. No mitochondrial deposits were observed when the temperatures of the fixatives rose to 37 and 55°C. These results indicated that peroxidase was very resistant to the heat with MWI fixation. Cytochrome oxidase is sensitive to the heat with MWI, so, a chilled water bath had to be used.