[Histonet] RE: endogenous peroxidases

[Tony Henwood]

An interesting article by Chun Gao,  Ai-Ying Wang, and Ya-Jing Han (Appl Immunohistochem Mol Morphol 2008;16:393-399) concluded that blockage of endogenous peroxidase with H2O2 in immunostaining could be omitted and replaced by microwave oven heating (using 0.01M citric acid buffer (pH 6.0)).

Regards 
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) 
Laboratory Manager & Senior Scientist 
Tel: 612 9845 3306 
Fax: 612 9845 3318 
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Johnson, Teri
Sent: Saturday, 23 April 2011 2:51 AM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: [Histonet] Re: endogenous peroxidases

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


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