RE: Antigen Retrieval

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From:"Tarpley, John" <> (by way of histonet)
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You don't mention which antibody you do first, but here's a suggestion that
works for us. First, do the vimentin since it doesn't require any
pretreatment for your method. I'm assuming here that you're doing one
antibody with HRP and the second with Alk Phos. If not, you'll have to
adjust accordingly. Following this assumption you would detect the vimentin
using HRP with DAB. Following your wash steps after DAB do the antigen
retrieval. We use Citrate Buffer and DAB is not harmed by the retrieval,
enzyme, or acid step. Now do the BrdU stain and detect with Alk Phos. If you
want permanent sections you could use Vector Red as the chromagen. It has
the added advantage that it is fluorescent. Best of luck.

John Tarpley 15-2-B
Associate Scientist
Specialist Image Analysis & Immunohistochemistry
Amgen Inc
One Amgen Center Drive
Thousand Oaks, CA  91320

> ----------
> From: 	Bennett, Catherine (Katie)[]
> Sent: 	Thursday, December 23, 1999 8:51 AM
> To: 	'Histonet Server'
> Subject: 	Antigen Retrieval
> I realize not many histonetters may be online right now, but to those out
> there, I present my current dilemma.
> I am trying to do double immuno staining for BrdU and Vimentin in 10%
> neutral buffered formalin-fixed rat lung sections.  I can get great BrdU
> labeling with almost all of the microwave antigen retrieval procedures
> I've
> been using (1% Zinc Sulfate, 0.01M Citrate Buffer, 0.1MTris-HCl, and 1mM
> EDTA).  But the Vimentin stain after microwave AR is really weak.  Without
> antigen retrieval, I get no BrdU staining but awesome Vimentin stains.  My
> protocol always includes a 5 minute 0.05% Protease K enzyme digestion
> step,
> and a 1h HCL denaturation step.  Without the microwave AR, these alone
> won't
> do the trick for the BrdU.
> I'm guessing my next step is to ditch the microwave AR since it kills the
> vimentin and play around with various enzyme digestion times to boost the
> BrdU.
> Anyone have any insight or suggestions?
> ****************************
> Catherine "Katie" Bresee Bennett
> Sr. Technical Associate
> Lovelace Respiratory Research Institute
> Albuquerque, New Mexico

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