Horseradish peroxidase (HRP) and alkaline phosphatase (AP) as referenced in the following images and antibody descriptions are most commonly used in enzymatic immunohistochemistry (IHC).
HRP is a 40 kD enzyme derived from the root of the horseradish plant. HRP enzyme reacts with chromogenic substrate 3, 3’ diaminobenzidine tetrahydrochloride (DAB) giving a brown end color reaction.
Red blood cells (RBCs) and some leukocytes contain endogenous peroxidase and will react with DAB unless quenched of this naturally occurring endogenous peroxidase activity. This is performed by application of hydrogen peroxide (H2O2) somewhere in the IHC protocol prior to DAB application. Typically, tissue sections are immersed in or a solution of H2O2 is applied to the tissue sections following paraffin removal, clearing, and hydration to water. Blocking of endogenous peroxidase activity can be performed prior to or after tissue pretreatment.
DAB reaction occurs when iron containing heme groups of peroxidase and H2O2 form a complex producing water and atomic oxygen. This complex oxidizes DAB (an electron dense donor), producing a stable brown color end product at the antigen-antibody complex. Today’s market offers DAB in an easy to use, two part product consisting of concentrated DAB chromogen and buffer substrate/H2O2 solution. The DAB working solution ratio is usually one drop of DAB concentrate to 1.0 mL of buffer substrate. The reaction time for working DAB is generally 5-10 minutes and DAB is more stable than fast red. Proper handling and safety guidelines MUST be adhered to when using DAB products, as it is classified as a suspected carcinogenic.DAB Enhancement
Solutions of metal compounds can be used to enhance DAB reactions. After washing tissue sections treated with DAB, the sections can be immersed in 1% solution of copper sulfate (prepared in distilled water) for 20-30 seconds to enhance the DAB reaction.
Alkaline phosphatase (AP) is derived from calf intestine and is used as an enzyme label in order to generate an alternate or contrasting color to the brown signal generated by DAB. A blocking solution or substrate buffer containing levamisole is required to prevent endogenous AP activity from occurring. However, tissues such as placenta and intestine containing AP isoenzymes may exhibit some background staining.