[Histonet] Endogenous Peroxide Treatment - Immunohistochemistry
anh2006 <@t> med.cornell.edu
Sun Mar 16 10:48:23 CDT 2008
I agree with Isaac, that the likely culprit when staining liver is
biotin background. I don't know whether that is or is not the issue
in your case until we knew more about your protocol.
For my protocols, I use 3% H202 in H20 for 10 min RT and it blocks
endogenous peroxidase in liver very well for most applications (the
endogenous biotin still needs to be blocked with a kit).
However, if there is extramedullary hematopoiesis or a large
infiltration of neutrophils you may see WBC associated endogenous
peroxidase which is nearly impossible to block with standard methods.
For this we use glucose oxidase blocking method. I have an excellent
protocol from Gayle Callis I have used for years I can share if you
like. In fact, Gayle may even respond to this email with the protocol
>If you are running your IHC stains with an HRP detection kit with a
>biotinylated secondary antibody, endogenous biotin within the tissue can
>cause background staining to occur. Often times, you'll see endogenous
>biotin in a tissue that is particularly bloody, such as liver, kidney,
>You can use an avidin/biotin block before secondary antibody is applied
>to account for this. You can find this block through many different
>companies - mine (Cell Marque) offers a good version. The part number
>is CMX222 and it can be ordered at 1-800-665-7284.
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Marilyn
>Sent: Saturday, March 15, 2008 11:27 AM
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] Endogenous Peroxide Treatment - Immunohistochemistry
>For routine tissues, I use 3% hydrogen peroxide for 10 mins. to remove
>Presently, I am trying to stain liver tissue, which stores more of the
>There seems to be background staining that masks blood cells and
>I've tried using 5% hydrogen peroxide solution for 10 mins. and there is
>Are there other solutions that can be used instead of hydrogen peroxide?
>Any replies would be greatly appreciated.
>Thank you in advance.
>Food and Safety Division
>Edmonton, Alberta, Canada.
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