RE: signal amplification methods

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From:"Sebree Linda A." <> (by way of histonet)
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Hi Yuhui,

We use Ventana automated instruments so we have the option of using their
Amplification Kit.  This is comprised of RxM IgG followed by MxR IgG. This
is applied after the primary and before the secondary.  In essence, you are
amplifying the binding sites of the primary antibody.  If this is not an
option, you can achieve similar results by making your own reagents.  We use
RxM IgG (Pierce, cat. #31190) at a 1:200 dilution in between the primary and
secondary antibodies (you could probably follow this with a MxR IgG although
we haven't tried this).  This is sometimes slightly dirtier than the Ventana
Amplification Kit but its a heck of a lot cheaper!  And one could certainly
play with the dilutions and incubation times to clean it up.  Unfortunately,
you are amplifying any endogenous peroxidase and/or biotin not just the
primary antibody sites, so you should make sure you have a nice clean stain
before you amplify. Obviously these methods are for use with monoclonal
antibodies although we have tried both our home brew amplification and
Ventana's with polyclonal antibodies and sometimes they do intensify the

Hope this is of some use,

Linda A. Sebree, HT
University of Wisconsin Hospital & Clinics
Immunohistochemistry/In Situ Hybridization Laboratory
600 Highland Avenue
Madison, WI  53792-2472

FAX: (608)263-1568

> -----Original Message-----
> From:	Yuhui_Xu []
> Sent:	Wednesday, November 10, 1999 7:56 AM
> To:
> Subject:	signal amplification methods
> Dear All:
> I understand that currently there have been methods to amplify
> immunohistochemical signals, e.g.,TSA by DuPont. I would appreciate
> information
> about other methods in this direction.
> Thanks in advance.
> Yuhui

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