Re: [Histonet] HRP-labeled primary antibodies
If there is enough of the antigen in the tissue or sample you can detect without amplification. HRP/DAB is an enzymatic reaction so it is also is amplification. I would try it the straight up way before diving into more complex protocols. Alternatively another option would be that you could try to come in with a secondary antibody (either HRP labeled itself or biotinylated). If your secondary is a polyclonal - which most are - it should still be able to detect the primary even with the HRP attached. Worth trying anyway. -Original Message- From: Kim Merriam kmerriam2...@yahoo.com Date: Thu, 21 May 2009 05:41:44 To: Histonethistonet@lists.utsouthwestern.edu; ih...@googlegroups.com Subject: [Histonet] HRP-labeled primary antibodies Hi All, Has anyone had experience doing IHC on FFPE tissues with HRP-labeled primary antibodies? I was wondering what the best way to detect them would be. I assume that going strait to DAB would not work, since no amplification is there. I was thinking of using a biotinyl tyramide step to amplify the signal. Also, do you think the final antibody concentration would need to be higher than with traditional, unlabeled primaries? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ 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] HRP-labeled primary antibodies
I got some great ideas. Thanks everyone! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA From: anh2...@med.cornell.edu anh2...@med.cornell.edu To: Kim Merriam kmerriam2...@yahoo.com; Histonet histonet@lists.utsouthwestern.edu; ih...@googlegroups.com Sent: Thursday, May 21, 2009 10:48:15 AM Subject: Re: [Histonet] HRP-labeled primary antibodies If there is enough of the antigen in the tissue or sample you can detect without amplification. HRP/DAB is an enzymatic reaction so it is also is amplification. I would try it the straight up way before diving into more complex protocols. Alternatively another option would be that you could try to come in with a secondary antibody (either HRP labeled itself or biotinylated). If your secondary is a polyclonal - which most are - it should still be able to detect the primary even with the HRP attached. Worth trying anyway. -Original Message- From: Kim Merriam kmerriam2...@yahoo.com Date: Thu, 21 May 2009 05:41:44 To: Histonethistonet@lists.utsouthwestern.edu; ih...@googlegroups.com Subject: [Histonet] HRP-labeled primary antibodies Hi All, Has anyone had experience doing IHC on FFPE tissues with HRP-labeled primary antibodies? I was wondering what the best way to detect them would be. I assume that going strait to DAB would not work, since no amplification is there. I was thinking of using a biotinyl tyramide step to amplify the signal. Also, do you think the final antibody concentration would need to be higher than with traditional, unlabeled primaries? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ 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: [IHCRG] Re: [Histonet] HRP-labeled primary antibodies
I agree with Kim, if I was worried about a weak signal using abs directly conjugated to hrp I would just ignore the fact that they have the hrp and use a detection such as labeled polymer matched to the species of the primary ab just like I would without the direct hrp conjugation, there should still be enough sites left on the primary antibody for a secondary detection reagent to attach to the species ab even with some being taken up by the hrp. Patsy Patsy Ruegg, HT(ASCP)QIHC IHCtech, LLC Fitzsimmons BioScience Park 12635 Montview Blvd. Suite 215 Aurora, CO 80010 P-720-859-4060 F-720-859-4110 wk email pru...@ihctech.net web site www.ihctech.net This email is confidential and intended solely for the use of the Person(s) ('the intended recipient') to whom it was addressed. Any views or opinions presented are solely those of the author. It may contain information that is privileged confidential within the meaning of applicable law. Accordingly any dissemination, distribution, copying, or other use of this message, or any of its contents, by any person other than the intended recipient may constitute a breach of civil or criminal law and is strictly prohibited. If you are NOT the intended recipient please contact the sender and dispose of this e-mail as soon as possible. _ From: ih...@googlegroups.com [mailto:ih...@googlegroups.com] On Behalf Of Kim Merriam Sent: Thursday, May 21, 2009 8:52 AM To: Histonet; ih...@googlegroups.com Subject: [IHCRG] Re: [Histonet] HRP-labeled primary antibodies I got some great ideas. Thanks everyone! Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA _ From: anh2...@med.cornell.edu anh2...@med.cornell.edu To: Kim Merriam kmerriam2...@yahoo.com; Histonet histonet@lists.utsouthwestern.edu; ih...@googlegroups.com Sent: Thursday, May 21, 2009 10:48:15 AM Subject: Re: [Histonet] HRP-labeled primary antibodies If there is enough of the antigen in the tissue or sample you can detect without amplification. HRP/DAB is an enzymatic reaction so it is also is amplification. I would try it the straight up way before diving into more complex protocols. Alternatively another option would be that you could try to come in with a secondary antibody (either HRP labeled itself or biotinylated). If your secondary is a polyclonal - which most are - it should still be able to detect the primary even with the HRP attached. Worth trying anyway. -Original Message- From: Kim Merriam kmerriam2...@yahoo.com Date: Thu, 21 May 2009 05:41:44 To: Histonethistonet@lists.utsouthwestern.edu; ih...@googlegroups.com Subject: [Histonet] HRP-labeled primary antibodies Hi All, Has anyone had experience doing IHC on FFPE tissues with HRP-labeled primary antibodies? I was wondering what the best way to detect them would be. I assume that going strait to DAB would not work, since no amplification is there. I was thinking of using a biotinyl tyramide step to amplify the signal. Also, do you think the final antibody concentration would need to be higher than with traditional, unlabeled primaries? Thanks, Kim Kim Merriam, MA, HT(ASCP)QIHC Cambridge, MA ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet http://lists.utsouthwestern.edu/mailman/listinfo/histonet ___ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet