Description
The application of antibodies and other proteins covalently coupled to horseradish peroxidase (HRP) in immunohistochemistry is well documented. In the presence of hydrogen peroxide (H2O2), HRP catalyzes the oxidation of TMB forming a blue chromogenic product visible by light microscopy. Researchers have shown that the reaction of TMB with HRP is a sensitive method for demonstration of retrograde tracing in neural tissue. Lu and Ho obtained excellent staining with TMB-molybdate in anterograde labeling of neural tissue at the ultrastructure level. When used in double-labeling for localization of innervations of feline cerebral arteries, TMB and DAB peroxidase substrates provided high resolution morphological results. Previous methods to stabilize the blue reaction color required multiple staining steps, but RealBlue provides permanent blue color when used as recommended. Like DAB, the RealBlue reaction product is insoluble in alcohols and Xylene. Endogenous peroxidase activity is eliminated by controlled oxidation prior to application of antibodies
