[62] Histochemical methods for localization of endothelial superoxide and hydrogen peroxide generation in perfused organs

摘要: Publisher Summary This chapter describes two histochemical techniques capable of revealing discrete sources biological oxidants in perfused organs at the light or electron microscopic levels. The first is a high manganese/diaminobenzidine technique, which superoxide oxidizes Mn 2+ to 3+ , turn diaminobenzidine (DAB) form amber-colored, osmiophilic polymers, observable by microscopy. second iron/diaminobenzidine hydrogen peroxide diethylenetriaminepentaacetate-chelated Fe intermediate species, oxidize DAB similarly . Both manganese and iron methods can readily demonstrate appearance reaction product on luminal surfaces arterial, capillary, venular endothelial cells during 2 3 rain reoxygenation after ischemia. reactions are nearly absent non-manganese- noniron-treated controls. Superoxide dismutase strongly inhibits /DAB reaction, catalase when tested specially lung preparations these specific antioxidant enzymes concentrated. These provide direct, visual evidence that burst reactive oxygen species generated isolated cultured tissues, using simple laboratory procedures available almost any investigator marginal cost.