Blocking Endogenous Peroxidase
Why should I block endogenous peroxidase acitivity
Some cells or tissues contain endogenous peroxidase. Using HRP conjugated antibody may result in high, non-specific background staining. This non-specific background can be significantly reduced by pre-treatment of cells/tissues with hydrogen peroxide prior to incubation with HRP conjugated antibody.
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 (H2O2), 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
Hydrogen peroxide should be stored in the refrigerator and protected from sunlight in order to slow it’s thermal decomposition. Never return unused hydrogen peroxide to the original container. Do not pipette from a reagent bottle of 30% or greater of hydrogen peroxide. 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. Therefore, methanol is a better choice in this case. Some cell surface markers are very sensitive to methanol/hydrogen peroxide quenching, reducing the staining of antigenic site, particularly on frozen sections. So using hydrogen peroxide in PBS is recommended for cell surface or membrane markers.
What concentration of hydrogen peroxide is commonly used
3% hydrogen peroxide is commonly used to block endogenous peroxidase activity. However, certain tissues/cells/antigen (i.e. cell surface markers such as CD4) can be destroyed by high concentration of hydrogen peroxide. So a lower concentration (0.3%) should be used.
Where should I do hydrogen peroxide blocking during IHC procedure
The blocking can be done (1) after rehydration to water and before antigen retrieval, (2) after antigen retrieval and before primary antibody incubation, (3) after primary antibody incubation, or (4) after biotinylated secondary antibody incubation. For certain antigens such as CD4 and CD8, hydrogen peroxide blocking has detrimental effect on the epitopes, thus reduce intensity of antibody staining. Therefore, blocking after primary antibody or secondary antibody incubation is recommended.
For how long the sections should be incubated in blocking solution
It can be 5 to 60 minutes depending on concentration of hydrogen peroxide, diluting reagent, tissue type, and fixation. 10-15 minutes incubation is commonly used for formalin-fixed, paraffin embedded tissue sections.
I blocked with hydrogen peroxide, but still getting background staining
The hydrogen peroxide may be expired. Try a fresh bottle of hydrogen peroxide and it may solve the problem. In addition, the tissue may contain endogenous biotin as well, so avidin/biotin blocking step may be needed. Finally, if the background problem still persists, you may consider switching to a different detection system, such as AP system or immunofluorescence method.
How to block endogenous peroxidase activity on frozen sections
Immerse slides in fresh made 0.3% hydrogen peroxide in 0.1% sodium azide for 10-15 minutes (to make the blocking solution, add 5ml of 3% hydrogen peroxide to 45 ml of 0.1% sodium azide and mix well). An alternative is to use 0.3% hydrogen peroxide in methanol for 20-30 minutes since methanol accelerates the destruction of the heme groups so a lower concentration of hydrogen peroxide can be used for longer incubation time.
What are alternative methods for blocking endogenous peroxidase activity
One alternative is to incubate sections in 0.36% beta-glucose-0.01% glucose oxidase-0.013% sodium azide in PBS for 60 minutes at 37 C. Another method is to incubate sections in 0.01% periodic acid for 10 minutes followed by 0.01% sodium borohydride in water for 2 minutes.