[Histonet] Re: troubles with CD4

[Amos Brooks]

Tom,
	What is the pH of the retrieval solution that you are using? In our experience CD4 tends to work best with a high pH retrieval solution (like Dako's high pH retrieval solution or Biocare Medical's Borg Decloaker).
	We use Sigma's Impress for detection of this because of it being a polymer which does not use avidin/ biotin it avoids the endogenous biotin background that high pH retrieval tends to increase.

Just some suggestions to try,
Amos Brooks

Message: 15
Date: Fri, 05 Aug 2005 10:47:22 -0500
From: "Thomas Pier" <tp2 <@t> medicine.wisc.edu>
Subject: [Histonet] troubles with CD4
To: <histonet <@t> lists.utsouthwestern.edu>
Message-ID: <s2f343c7.086 <@t> gwmail.medicine.wisc.edu>
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Hello all,
I was wondering if anyone could help me with some problems that I'm having with staining for CD4.  My control tissue for the work up is formalin fixted, paraffin embeded tonsil.  I'm using Labvision's CD4 Ab-2 clone 1F6.  I have diluted it anywhere from 1:10-1:50.  I'm using 1% goat serum in TBS as my diluent.  I've tried EDTA HIER as recomended on the data sheet.  I've also tried protease XXV w/ or w/o the EDTA.  I shouldn't be running into the peroxidase problem since I'm using an alkaline phosphatase detection system (Biocare's Vulcan Fast Red).  I have also tried using or not using a 10% goat serum protein block.  I have gotten some staining, but nowhere near as much as I should in a tonsil.  Any help that you could give me would be greatly appreciated.

Tom Pier