True the 4b12 cd4 clone performs better in paraffin, still as you say, cd4
is just not the most robust ab in my hands either, one thing I have found
(actually I need to credit Bryan Hewlett for this), is that you need to do
endogenous h202 block after hier and you should use h202 in methanol,
perhaps it is something about the methanol fixing after hier for ar that
helps with cd4.
Patsy

Patsy Ruegg, HT(ASCP)QIHC
IHCtech
12635 Montview Blvd. #215
Aurora, CO 80045
720-859-4060
fax 720-859-4110
pruegg@ihctech.net
www.ihctech.net
www.ihcrg.org    
 


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu
[mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Milne, Katy
Sent: Friday, November 07, 2008 9:21 AM
To: histonet@lists.utsouthwestern.edu; pieronelva01@bigpond.com
Subject: [Histonet] RE:CD4 on Ventana

Hi Piero, what clone are you using for CD4?

There seem to be 2 main clones, 1F6 or something like that and 4B12.
The epitope recognized by 1F6 doesn't do well in formalin so 4B12 is the
one most used.  Having said that, I've found it's still not wonderful, I
can get staining (I use a Ventana Discovery) but because the Ab is
recommended to be used at such a high concentration, you do have to pick
out the real staining against some background.

I used it in this paper, our general protocol is in there too but I'm
not sure how much the benchmark differs from the discovery....
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.00034
09

Katy Milne 
Deeley Research Centre

Message: 19
Date: Fri, 7 Nov 2008 20:15:11 +1100
From: "Piero Nelva" 
Subject: [Histonet] CD4 on Ventana
To: 
Message-ID: <2D0669F5E5EA4786A17AE07D652FFF51@pentium4>
Content-Type: text/plain; format=flowed; charset="iso-8859-1";
	reply-type=original

Dear Histonetters

I'm having a lot of trouble getting CD4 to stain FFPE tissue using the 
Ventana Benchmark XT.  Any advice about which antibody and protocol 
suggestions would be greatly appreciated.

Piero Nelva
Anatomical Pathology
Monash Medical Centre
Australia 


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