Enzyme Labels

Horseradish peroxidase (HRP) and alkaline phosphatase (AP), as referenced in the antibody descriptions and images that follow in this course, 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 applying hydrogen peroxide (H₂O₂) 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 deparaffinization, 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 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.

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% copper sulfate (prepared in distilled water) for 20–30 seconds to enhance the DAB reaction.

Alkaline phosphatase (AP) is derived from calf intestine. It is used as an enzyme label 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 the placenta and intestine containing AP isoenzymes may exhibit some background staining.