[Histonet] CD4 & CD8

From:Amos Brooks

Liz & Gayle,
         Actually we've had really good luck with both CD4 & CD8. The trick 
with CD4 is doing a peroxidase block before retrieval or not at all. If you 
do it after retrieval it will really diminish the reactivity. Use high pH 
epitope retrieval (I'm using DAKO for this in a steamer for 20 min). You 
should also use a non-biotin secondary antibody as the retrieval really 
brings out the endogenous biotin. Try Impress from Vector it's great! For a 
primary antibody we also use Neomarkers for both. Try around 1:10 - 1:20 
for a dilution.

Best of luck,
Amos Brooks

At 11:54 AM 1/11/2005, you wrote:
>Message: 8
>Date: Mon, 10 Jan 2005 15:00:52 -0700
>From: Gayle Callis 
>Subject: Re: [Histonet] CD21, B220, CD4
>To: "Elizabeth Chlipala" ,
>         Histonet@lists.utsouthwestern.edu
>         <>
>Content-Type: text/plain; charset="us-ascii"; format=flowed
>As discussed many times on Histonet, CD4 will NEVER work on FFPE!  FYI, CD8
>also will not work.
>If you have to run all these antibodies on the same sample, you will have
>to do frozen sections in order to accomadate the CD4.  FS are air dried
>overnight at RT over dessicant.   To improve morphology, use 75%
>acetone/25% absolute ethanol (no substitute on alcohol here) for 5 min at
>RT next day.  Do NOT air dry again, go directly from fixative to buffer and
>then stain.  We actually use biotinylated primaries more than purified,
>then come back with Strepavidin-HRP, then DAKO AEC+ or Chris van der Loos
>AEC - his formulation is excellent inhouse chromogen.  Using biotinylated
>primaries shortens a protocol!
>We buy all antibodies, monoclonals from BD PharminGen.  SEROTEC is another
>supplier.  These are rat antimouse monoclonals.

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