[IHCRG] Re: [Histonet] HRP-labeled primary antibodies

[Patsy Ruegg]

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 pruegg <@t> ihctech.net
web site www.ihctech.net

 


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From: ihcrg <@t> googlegroups.com [mailto:ihcrg <@t> googlegroups.com] On Behalf Of
Kim Merriam
Sent: Thursday, May 21, 2009 8:52 AM
To: Histonet; ihcrg <@t> 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: "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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