[Histonet] IHC and IF

[ti fei]

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"<kmerriam2003 <@t> yahoo.com>;
 Date:  Thu, Apr 15, 2010 07:38 PM
 To:  "Thotakura, Anil Kumar"<a.thotakura <@t> imperial.ac.uk>; "Histonet"<histonet <@t> 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.thotakura <@t> imperial.ac.uk>
To: "Histonet <@t> lists.utsouthwestern.edu" <Histonet <@t> 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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