Cytochrome oxidase (CO) is considered to be an enzyme inherent in the mitochondrial membrane, and is highly active in cells containing a large number of mitochondria (e.g., myocardium, renal tubular epithelium, as well as gastric lining cells, and hepatocytes), and the activity of this enzyme is often used as an indicator of oxidative metabolism within the cell, and as one of the hallmark enzymes of mitochondria.

Cytochrome oxidase staining solution (benzidine method) is based on the principle that cytochrome oxidase catalyzes the oxidation of 3,3-diaminobenzidine (DAB) to oxidize the amino group of the side chain, and then undergoes repeated oxidative polymerization and oxidative cyclization to form the insoluble brown phenazine polymer, which is sensitive to the fixative, so it is necessary to use fresh slices.