[Histonet] More on H202 issues

[Lewis, Patrick]

Hi Everyone

Thanks for your responses.

I am looking at cell surface markers,
Sorry I should have said.

Based on what I found out below:

Methanol is out, even though I agree that Methanol does enhance the effect of H202 blocking.
(I suppose I could try it to see how much/if any epitope loss there is in relation to H202 quenching,  At least It would help identifying false positives that are actually H202 artifacts.)

Also it looks like increasing the concentration of H202 is out.

As to when though,

It looks like with cell surface markers I should block after the primary, or even after the 2ndary?
Can I do that successfully with a HRP labeled 2ndary?

thoughts?

Patrick.


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.


Patrick Lewis
Research Associate II Bench
Seattle Childrens Research Institute
206-884-1115

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