hi if u use triton for permeablization in IHC
it sometimes cause the stain pattern to diffuse from nucleus to cytoplasm
it is common
------------------ Original ------------------
From: "Kim Merriam"<kmerriam2...@yahoo.com>;
Date: Thu, Apr 15, 2010 07:38 PM
To: "Thotakura, Anil Kumar"<a.thotak...@imperial.ac.uk>;
"Histonet"<histonet@lists.utsouthwestern.edu>;
Subject: Re: [Histonet] IHC and IF
Hi Anil,
Perhaps you are having mouse-on-mouse issues with your IHC (DAB) and you are
seeing antibody cross-reactivity with mouse IgGs (you don't mention your IHC
detection method, so I could be wrong). You are probably not seeing this
cross-reactivity with the IF method, because the labeling is direct. If that
is the case, I would be more likely to trust what I saw with the
directly-labeled antibody.
If you have labeled the monoclonal antibody with FITC and you want to see it
with a chromagen, you could use an anti-FITC secondary (Invitrogen makes a nice
Rabbit anti-FITC) which you could then detect with an anti-Rabbit polymer (or
whatever you normally use to detect a rabbit primary).
Good luck,
Kim
Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA
________________________________
From: "Thotakura, Anil Kumar" <a.thotak...@imperial.ac.uk>
To: "Histonet@lists.utsouthwestern.edu" <Histonet@lists.utsouthwestern.edu>
Sent: Thu, April 15, 2010 7:14:26 AM
Subject: [Histonet] IHC and IF
Dear All,
I have kind of funny problem.
I am doing IHC (substrate reaction suing Dabs reagent) and IF ( direct
labeling of my monoclonal antibody with flurochrome).
I am working on mouse liver frozen sections. When I did IHC the staining
looks like cytoplasm and if I do IF for the same protein I saw nuclear
staining. I am unable to conclude whether it is cytoplasm or nuclei
staining.
Please advice. The monoclonal antibody I used is not commercially available.
We made it.
Thanks In advance.
Many Thanks,
Anil Kumar.
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