Immunohistochemistry (IHC) commonly uses reagents that are found in cells, such as biotin and certain enzymes, which can interfere with antigen detection by generating unwanted signals that falsely indicate the presence of an antigen. Therefore, steps must be taken to determine if endogenous factors will interfere with signal detection and then prevent their interference in the detection of the target antigen.

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Endogenous Biotin

Biotin is a coenzyme in many biological reactions that is conjugated to antibodies and enzymes for labeling purposes based on a strong binding affinity with avidin.

Avidin-Biotin Complex methods are commonly used in IHC to enhance the detection of a target antigen. Because free or labeled avidin is used in these approaches, tissues rich in endogenous biotin, including liver, mammary gland, adipose tissue and kidney, can show high background staining because the avidin binds to the endogenous biotin (Dakshinamurti, K. et al., 1963). Endogenous avidin activity is most pronounced in cryostat sections.

For avidin-biotin-conjugation IHC detection systems, endogenous biotin should be blocked to avoid recognition of endogenous biotin along with the target antigen. The basic procedure is as follows:

  1. Coat the sample with an excess of free avidin.
  2. Add an excess of free biotin to fill all biotin-binding sites in the tetrameric avidin molecules (Wood, G.S. et al., 1981)

The end result is that all endogenous biotin is bound to avidin, and all biotin-binding sites on each avidin molecule are filled. Because HIER may increase the level of endogenous biotin that can be detected in a sample, negative control samples should also undergo HIER to reduce the possibility of false positives from increased biotin detection.

Avidin is highly glycosylated, and these carbohydrates can bind to sugar-binding lectins in tissues and increase the background signal. One approach to preventing this interaction is to use an analog of the avidin carbohydrate to saturate these binding sites on the tissue sample (Naritoku, W.Y. et al., 1982). Alternatively, replacing avidin with unglycosylated streptavidin or NeutrAvidin Protein, a deglycosylated form of avidin, will also eliminate this interaction.

Chromogenic IHC detection of cytokeratin 18 in colon carcinoma tissue. Endogenous peroxidase was quenched with Thermo Scientific Pierce Peroxidase Suppressor and endogenous biotin was blocked prior to incubating the sample with biotinylated cytokeratin 18 antibody. Cytokeratin 18 was detected with Thermo Scientific NeutrAvidin High Sensitivity HRP Conjugate in combination with Thermo Scientific Pierce Metal-Enhanced DAB. Cell nuclei were counterstained with hematoxylin.


Peroxidases, most often horseradish peroxidase (HRP), is commonly used as the reporter for target antigen detection by IHC. Final analysis of this staining method is complicated by the presence of endogenous peroxidase and "pseudoperoxidase" activity in the cells and tissues. Endogenous peroxidase reacts with hydrogen peroxide to reduce the 3,3'-diaminobenzidine (DAB) substrate or other peroxidase substrates, resulting in nonspecific staining of the tissue.

To confirm the presence of endogenous peroxidase activity, the fixed tissue should be reacted with a peroxidase substrate such as DAB. Any colored precipitate that forms in the tissue from this treatment indicates endogenous activity. If a tissue is rich in peroxidase activity, an alternative enzyme label, such as calf intestinal alkaline phosphatase (AP), can be used.

Several methods have been devised to inhibit or destroy endogenous peroxidase activity after tissue fixation. The most common reagent is 3% H2O2 in methanol or water (Streefkerk, J.G. et al., 1972), although commercial peroxidase suppressors are available that quench peroxidase activity more effectively. To ensure that the peroxidase suppressor does not affect the immunoreactivity of the primary antibody, the suppressor should be applied after incubation with the primary antibody and before incubation with the peroxidase conjugate (Fink, B. et al., 1979).


Phosphatases hydrolyze phosphate groups on cellular substrates, and calf intestinal alkaline phosphatase (AP) is commonly used as a reporter for IHC detection. Endogenous phosphatases can react with the IHC substrate, a combination of nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP), resulting in nonspecific staining.

A similar test, as described for peroxidases, can be employed using NBT/BCIP to determine if endogenous phosphatases need to be quenched. If they do, then the phosphatase inhibitor levamisole can be added between the primary and secondary antibody incubation steps to inhibit endogenous phosphatase activity.


  1. Dakshinamurti, K. and Mistry, S.P. (1963) J. Biol. Chem. 238, 294.
  2. Naritoku, W.Y. and Taylor, C.R. (1982) J. Histochem. Cytochem. 30, 253-260.
  3. Streefkerk, J.G. (1972) Nature 330, 80.
  4. Fink, B. et al., (1979) J. Histochem. Cytochem. 27, 1299.
  5. Wood, G.S. and Warnke, R. (1981). J. Histochem. Cytochem. 29, 1196-1204.