[Histonet] HRP-labeled primary antibodies

[Kim Merriam]

I got some great ideas.  Thanks everyone!
Kim
 Kim Merriam, MA, HT(ASCP)QIHC
Cambridge, MA 




________________________________
From: "anh2006 <@t> med.cornell.edu" <anh2006 <@t> med.cornell.edu>
To: Kim Merriam <kmerriam2003 <@t> yahoo.com>; Histonet <histonet <@t> lists.utsouthwestern.edu>; ihcrg <@t> 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 <kmerriam2003 <@t> yahoo.com>

Date: Thu, 21 May 2009 05:41:44 
To: Histonet<histonet <@t> lists.utsouthwestern.edu>; <ihcrg <@t> 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 



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