[Histonet] Endogenous Peroxide Treatment - Immunohistochemistry

[Andrea Hooper]

Dear Marilyn,

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 
herself also.

Cheers,
Andrea



>Hi Marilyn,
>
>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,
>or brain.
>
>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.
>
>Happy staining!
>
>Isaac
>
>-----Original Message-----
>From: histonet-bounces <@t> lists.utsouthwestern.edu
>[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Marilyn
>Johnson
>Sent: Saturday, March 15, 2008 11:27 AM
>To: histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] Endogenous Peroxide Treatment - Immunohistochemistry
>
>Hi Histonetters,
>For routine tissues, I use 3% hydrogen peroxide for 10 mins. to remove
>RBC's.
>Presently, I am trying to stain liver tissue, which stores more of the
>blood components.
>There seems to be background staining that masks blood cells and
>vessels.
>I've tried using 5% hydrogen peroxide solution for 10 mins. and there is
>no improvement.
>Are there other solutions that can be used instead of hydrogen peroxide?
>Any replies would be greatly appreciated.
>Thank you in advance.
>
>Marilyn Johnson
>Alberta Agriculture
>Food and Safety Division
>Edmonton, Alberta, Canada.

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