Re: IHC in frozen mouse liver

From:Gayle Callis

The DAKO Envision kit avoids avidin/biotin systems, and is very sensitive.
You would still have to use Peroxidase blocking, but DAKO also has a nice
endogenous peroxidase blocker for frozen sections.  They also have a
nonprotein blocker that works also. Saw this in use in Chris van der Loos's
lab in Amsterdam, was impressed with results on polyclonal primary


At 07:22 AM 7/19/02 +0000, you wrote:
>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"
>MSN Photos is the easiest way to share and print your photos: 
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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