Why block endogenous peroxidase activity

Using HRP conjugated antibody may result in high, non-specific background staining. How do I know if my tissues contain endogenous peroxidase activity. Incubate your tissue sections with DAB substrate solution after rehydration to water. If it turned brown, the tissue contains endogenous peroxidase and a blocking step is needed. What chemicals should I use to block endogenous peroxidase activity. Hydrogen peroxide H 2 O 2 , a blocking agent in immunohistochemistry, is commonly used to block endogenous peroxidase acitivity.

Pre-treatment with saturating amounts of hydrogen peroxide results in the irreversible inactivation of endogenous peroxidase. What should I know when handling hydrogen peroxide.

Never return unused hydrogen peroxide to the original container. If contaminated with certain metals or their salts, hydrogen peroxide can decompose violently. The bottle is vented because hydrogen peroxide decomposes in the presence of traces of impurities, yielding oxygen and water.

This could cause dangerously high pressure in a tightly capped container. What solutions or reagents should I use to dilute hydrogen peroxide. Methanol , PBS , distilled water or saline can be used to dilute hydrogen peroxide. Morphology of blood smears and peroxidase-rich tissues could be damaged by the aqueous hydrogen peroxide solution.

See the article " A possible role of thymidine phosphorylase expression and5-fluorouracil increased sensitivity in oropharyngeal cancer patients " on page This article has been cited by other articles in PMC. Dear Editor, In two recent JCMM papers dealing with the staining of various human tissue antigens, divergent approaches for blocking endogenous peroxidase activity were reported.

References 1. A possible role of thymidine phosphorylase expression and 5- fluorouracil increased sensitivity in oropharyngeal cancer patients. Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry. Inhibin is not an immunohistochemical marker for hepatocellular carcinoma. An example of the potential pitfall in diagnostic immunohistochemistry caused by endogenous biotin. Am J Clin Pathol. The enhanced reactivity of endogenous biotin-like molecules by antigen retrieval procedures and signal amplification with tyramine.

Histochem J. Boenisch T. Handbook of immunochemical staining methods. Reduced hippocampal insulin-degrading enzyme in late-onset Alzheimers disease is associated with the apolipoprotein E-e4 allele. They will create pores large enough for antibodies to pass through without dissolving the plasma membrane. They are suitable for antigens in the cytoplasm or the cytoplasmic face of the plasma membrane, and are also suitable for soluble nuclear antigens.

This non-specific background can be significantly reduced by pre-treatment of the sample with hydrogen peroxide before incubation with HRP-conjugated antibody.

Morphology of blood smears and peroxidase-rich tissues can sometimes be damaged by the hydrogen peroxide. Diluting the hydrogen peroxide in methanol is the best choice for fragile samples where preservation of morphology is required. However, some cell surface protein markers are very sensitive to methanol or hydrogen peroxide quenching, reducing the staining of antigenic site, particularly on frozen sections. Using hydrogen peroxide in PBS is suggested for cell surface or membrane proteins.

Another protocol modification is to quench with peroxide after first incubating section or cells with the primary antibody. The fixation and permeabilization method used will depend on the epitope and the sensitivity of the antibody, and may require some optimization.

Fixation can be done using crosslinking reagents, such as paraformaldehyde. These are better at preserving cell structure, but may reduce the antigenicity of some cell components, as the crosslinking will obstruct antibody binding antigen retrieval techniques may be required.

Another option is to use organic solvents such as methanol, ethanol and acetone. These remove lipids while dehydrating the cells. They also precipitate proteins on the cellular architecture. This will leave a small circle of cell sample adhered to the centre of the slide which can be dried, fixed and stained. A list of fluorochromes and their excitation and emission wavelengths can be found here.

In order to be able to examine the co-distribution of two or more different antigens in the same sample, a double or triple immunofluorescence or immunohistochemistry procedure can be carried out. Primary antibodies against two or three different target proteins can be used either in parallel in a mixture or sequentially.