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. _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
