Re: IHC in frozen mouse liver

From:louise renton

DEar John,
Liver contains HUGE amounts of biotin which would react with the avidin in 
the ABC kit, use another detection system such as peroxidase/antiperoxidase 
(PAP) or glucose oxidase. This will clear up  the background. One of the 
snags using PAP however, is loss of signal, but this may not be a problem if 
you are using frozen tissue. Try using an endothelial marker as a control 
(CD34 springs to mind, but I don't know if there is a polyclonal available. 
CEA might also also be an option.
Best regards
Louise renton

>From: John E Morales 
>To: HistoNet Server 
>Subject: IHC in frozen mouse liver
>Date: Thu, 18 Jul 2002 12:59:09 -0500
>I am trying to establish a protocol for immunohistochemistry in
>frozen mouse liver sections in our lab.  I would first like to know
>what antibody to use as a good positive control (for liver) to
>determine whether a procedure is working in current and/or future
>experiments. I would also like to ask anyone who has successfully
>performed such an experiment to email any protocols, sources of
>information or knowledge they may have on the subject.  I'll name my
>first born after you or give you a kidney.
>     Additionally, I have tried the Vector ABC detection kit only to
>recieve a high background (even after A/B blocking) and I have so far
>not had any trouble with endogenous peroxidases showing background(is
>this normal?).  I have recently tried IHC using a SRB1 primary antibody
>and detection with an HRP labelled secondary with the results as
>SRB1 (IgG from rabbit)= good signal
>Neg.control(No primary)= no signal, very clean
>Neg.control(serum IgG from a non-innoculated rabbit)= low signal
>Seeing as my non-innoculate negative control was not clean I am unsure
>about the results.  Suggestions?  Comments?  Thank you very much for
>any help and time you provide,
>Research Assistant II

Louise Renton
Bone Research Unit
South Africa
Tel & fax +27 11 717 2298
"Time flies like an arrow, friut flies like a banana"

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