Re: IH on Liver Tissue

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From:Phyllis Davie <>,
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  You actually need to block for 2 different things.  
     The first is endogenous peroxidase, which you are already blocking 
for.  (To see if it is effective, check the red blood cells--if they are 
brown, your peroxidase blocking is not working,  if they are clear, it 
     The second thing to block for is endogenous biotin (I am assuming 
you are using an avidin-biotin system).  Judging from your description of 
your tissue, you are looking at an endogenous biotin problem.  If the 
hepatocytes are all brown, your problem is far more likely to be biotin 
than peroxidase.  There is endogenous biotin in many tissues, but liver 
is particularly rich in it (so are kidney and brain).  There are many 
commercial kits available for blocking endogenous biotin.  There are also 
several techniques for making the reagents yourself (look at the 
recent/ongoing thread on the histonet). 
     We also have a problem with endogenous biotin.  It seems to be 
enhanced by the HIER (i.e. we see the problem more often, and more 
intensly in tissues that have HIER, than in those which have enzymatic 
digestions, or no pretreatment at all).  I would recommend that you do 
some form of biotin blocking.

Hope that helps,

Phyllis Davie

>How can you successfully perform immunohistochemistry on slides of liver
>tissue using horse-radish peroxidase and DAB?  How do you quench the
>endogenous peroxidase in the tissue so everything doesn't turn brown?  I've
>tried 3, 10 and 15 % H2O2 in both methanol and di water for 20 min. up to 1
>hour with no success.  The tissues are formalin fixed and embeded in
>paraffin.  We then used a pH 6.0 citrate buffer/microwave antigen retrieval
>system for our primary Ab detection.  Even with all that the endogenous
>enzyme caused the false positive with the DAB.  Anyone have any
>Thank you for all replies.
>Karen in Omaha

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