[Histonet] Re: endogenous peroxidases

[Johnson, Teri]

Hi Emily,

As for when is better to block endogenous peroxidases, I have no direct experience but I have heard from some that it should be done post-AR because the retrieval can reactivate the endogenous peroxidase. So that's when we do it.

Some folks worry about what effect that might have on their primary antibody binding with their target and will do their block after application of the primary antibody, prior to the secondary (or whichever step has the HRP label). I try to keep it simple and just do my H2O2 block prior to the protein blocking step.

Also, we never use methanol as a solvent for diluting the H2O2. It is contraindicated in some CD marker staining, and it's cheaper and just as good to use aqueous 3% H2O2. Some make it up in buffer, and you can do that as well if you wish. For cryosection peroxidase quenching, we use 0.3% H2O2 for 30 minutes. Otherwise it's a 10 minute step right after the AR in our routine paraffin section IHCs.

Hope this helps!

Teri

Teri Johnson, HT(ASCP)QIHC
Head, Histology and Electron Microscopy
Stowers Institute for Medical Research
Kansas City, MO