Reappraisal of potassium permanganate oxidation applied to Lowicryl K4M embedded tissues processed by high pressure freezing/freeze substitution, with special reference to differential staining of the zymogen granules of ratgastric chief cells

The high pressure freezing/freeze substitution technique is known to yield a deep vitreous freezing of tissues. Combination of this technique with Low icryl K4M embedding allows us histochemical studies of dynamic cellular pro cesses with improved structural preservation. The disadvantage of Lowicryl K4M embedding is its poor electron density in electron microscopy. To addre ss this problem, we examined the effects of KMnO4 oxidation applied to Lowi cryl K4M embedded rat gastric glands processed by high pressure freezing, T he KMnO4 oxidation-uranyl acetate-lead citrate sequence succeeded not only in contrast enhancement of cellular components, but also in differential st aining of the zymogen granules of rat gastric chief cells. This technique c ould be applied to semi-thin sections of Lowicryl K4M embedded rat gastric glands. The KMnO4 oxidation-toluidine blue staining provided sufficient con trast with regard to the zymogen granules. Various experiments used in this study verified that the KMnO4 oxidation plays an essential role in the dif ferential staining of the zymogen granules. Combined use of the KMnO4 oxida tion with phospholipase Ar-immunostaining demonstrated that gold labeling w as localized to the zymogen granules without the loss of immunolabeling. En ergy dispersive X-ray microanalysis revealed some manganese depositions on the zymogen granules. It is highly anticipated that the KMnO4 oxidation wil l become a useful tool for histochemical investigations combined with cryof ixation/freeze substitution and low temperature embedding techniques.