Cytochrome oxidase activity and confocal laser scanning microscopic analysis of the hamster submandibular gland using microwave irradiated fixation

Submandibular glands of the hamster were irradiated in 2% paraformaldehyde (pFA)-0.5% pure glutaraldehyde (PGA) with a microwave (MW) processor at temperatures of 10degrees and 37degreesC. Electron microscopy showed that cytochrome oxidase activity was taking place in the mitochondrial intermembrane-intracristal space of the granular duct cell when the temperature of the MW-irradiated fixatives was at 10degreesC. However, a decrease of this activity was observed when we took care to keep the temperature of the MW-irradiated fixatives at 37degreesC. The distinct reduction of cytochrome oxidase activity allowed by MW irradiation seems to be due the thermal affects of fixatives. Of course, the possibility cannot be excluded that MW irradiation caused other undetectable membrane damage. Then, we used confocal laser scanning microscopy for the preservation check of the mitochondrial membrane for cytochemistry with MW-irradiated fixation. The fluorescence of rhodamine 123 was observed in the inner spaces of the mitochondria at temperatures of 10degrees and 37degreesC. When the same tissues were fixed with 2% pFA using an MW processor as the sole fixative at 10degreesC, no mitochondrial fluorescence was observed. Cytochrome oxidase activity, by contrast, could be seen in the n-titochondrial intermembrane-intracristal spaces in the same condition. Formaldehyde is not the best aldehyde for the purpose of ultrastructural preservation. On the other hand, light and electron microscopy showed that the endogenous peroxidase activity was localized in the nuclear envelope, endoplasmic reticulum, secretory granules, and Golgi apparatus of the hamster submandibular gland using 2% pFA-0.5% PGA fixative with and without MW irradiations at temperatures of 10degrees and 37degreesC. Some of the same cells were fixed with only 2% pFA under MW irradiation at 10degreesC; however, marked diffuseness of the peroxidase activity was observed. Therefore, these results indicated that cytochrome oxidase activity was sensitive to heat with MW-irradiated fixation. Peroxidase activity was very resistant to heat with MW-irradiated fixation but not with pFA solo fixation, therefore, PGA had to be used.