Abstract:     Currently, many kinds of enzymes are applied in immunostaining. Horseradish peroxidase(HRP) and alkaline phosphatase(AP) are the two enzymes most widely used in enzyme labeling methods.
Keywords:  Immunohistochemistry, Enzymology, Horseradish Peroxidase(HRP), Alkaline Phosphatase(AP), Immunostaining

1. Horseradish Peroxidase(HRP)

The substrate of HRP is hydrogen peroxide. Peroxidation products oxidize chromogen and precipitate in the binding site of antibodies. 3,3'-Diaminobenzidine tetrahydrochloride (DAB) is the most commonly used chromogens,  which can produce brown precipitation insoluble in organic solvents. If the activity of endoperoxidase or melanin content is higher, DAB isn't the best choice. Then, other chromogens or alkaline phosphatase should be considered. Another chromogen of peroxidase 3-Amino-9-ethylcarbazole (AEC) produces red precipitation soluble in organic solvents and should be sealed in water-soluble liquid. Other chromogens can also produce similar red precipitation insoluble in organic solvents. 4-Chloro-1-naphthol can produce blue precipitation soluble in organic solvents. Currently, several different experimental methods can enhance the colour of DAB end products.

2. Alkaline Phosphatase(AP)

Alkaline phosphatase is separated from the intestinal tissue of calf. Basic principles are as follows. By hydrolyzing phosphorous salts containing naphthol substitutes, insoluble naphthol derivatives are produced. Then, the derivatives bind with suitable diazonium salts and produce colored insoluble azo dyes in the active site of the enzyme. The most frequently used chromogens are BCIP/NBT(blue, stable medium), Fast Red (red, water soluble sealing reagent) and New Fuchsin (magenta, water soluble sealing reagent). Alkaline phosphatase is suitable for immunocytochemistry test for cell samples or tissue samples with higher endoperoxidase. Alkaline phosphatase and horseradish peroxidase can be jointly applied in double immunohistochemical staining.

3. FineTest IHC Products

FineTest has rich experience in HRP enzyme labeling. Applications of HRP conjugated secondary antibodies in IHC staining are shown in the figures below.

Figure 1. Gastric cancer 20×.

Figure 2.  Kidney 10×.

Figure 3. Colon cancer.

Table 1. Selected FineTest IHC Products

Cat No.	Product Name
IHC0001	Fine Test SABC KIT
IHC0002	Fine Test SABC KIT
IHC0003	Fine Test SABC KIT
IHC0004	Fine Test SABC KIT
IHC0005	DAB Substrate Kit
IHC0007	Rabbit-DAB (Poly-HRP) Detection IHC Kit
IHC0008	Mouse-DAB (Poly-HRP) Detection IHC Kit

REFERENCES

[1] Horseradish peroxidase: a modern view of a classic enzyme.
[2] Alkaline Phosphatase: Discovery and Naming of Our Favorite Enzyme.
[3] Multiple Antigen Immunostaining Procedures.