RE: SPAM-LOW: Re: [Histonet] IHC and IF
I agree with Kim, your cytoplasmic staining with hrp/dab is probably because your anti mouse detection is reacting to endogenous Mouse IgG in the mouse tissue you are using. Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech 12635 Montview Blvd. Ste.215 Aurora, CO 80045 720-859-4060 fax 720-859-4110 www.ihctech.net www.ihcrg.org -Original Message- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Kim Merriam Sent: Thursday, April 15, 2010 5:39 AM To: Thotakura, Anil Kumar; Histonet Subject: SPAM-LOW: 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" To: "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 ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
Re: [Histonet] IHC and IF
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"; Date: Thu, Apr 15, 2010 07:38 PM To: "Thotakura, Anil Kumar"; "Histonet"; 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" To: "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___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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" To: "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
[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
