Dear Histonetters,
I've got many requests asking for our protocol for staining CD4 and  CD8, - therefore 
I think it is best to answer in general on the histonet for all users. Since I was out-
house for several days, I apologize for any delay in my answer !

We make pretreatment for CD8 and CD4 in a household microwave oven, place the 
slides well submerged in buffer (0,8 liter container) for 30 minutes, using 800 Watt.
For bone-marrow (decalified by EDTA in a ultrasonic bath), we use as buffer 
monocitrate pH 6.0.
For all regular human tissues, we use monocitric buffer pH 7.2

For CD4, we use Zymed "ready-to-use" monoclonal antibody 08-0101.
For CD8, we use Dako antibody (M7103) in a dilution 1:40

For both markers, detection is done with the Zymed detection kit "Zytochem plus 
HRP 5000".
All stainings are done in daily routine on a Lab-Vision Autostainer. Sorry, but we have 
only experience in human FFPE tissue. I have no idea if it works in bovine tissue as 
well. In mouse tissue, it doesn't work - that's why I posted my initial request to 
histonet to get protocols for mouse FFPE. All the answers I got hint to the fact that 
there are no reliable protocols or antibodies for CD4/8 in mouse FFPE so far. 
However, we will not give up and try harder to solve this dilemma - as soon as we 
have answers, I'll let you know !

Best regards - and thanks for the many answers and comments from you.
Have a successful new years 2006, - with less war on earth and more thruth.

Sincerely yours
Reinhard.





Date sent:      	Mon, 19 Dec 2005 19:10:42 +0100
From:           	histonet-request@lists.utsouthwestern.edu
Subject:        	Histonet Digest, Vol 25, Issue 24
To:             	histonet@lists.utsouthwestern.edu
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Today's Topics:

   1. CD4 and CD8 in mouse / human FFPE
      (wasielewski.reinhard.von@mh-hannover.de)
   2. Re: CD4 and CD8 in mouse / human FFPE (Laurie Reilly)


----------------------------------------------------------------------

Message: 1
Date: Sun, 18 Dec 2005 20:49:21 +0100
From: wasielewski.reinhard.von@mh-hannover.de
Subject: [Histonet] CD4 and CD8 in mouse / human FFPE
To: histonet@lists.utsouthwestern.edu
Message-ID: <43A5CB51.20138.3A8527E7@localhost>
Content-Type: text/plain; charset=US-ASCII

Hi Patsy,
we do a lot of CD4 and CD8 in human FFPE tissues, even in BM after decalification. 
It works very good and is daily routine in our lab. If you need advice, give me a mail.
We now got some hints how both markers could work in mouse tissue, too. If we 
succeed, we'll let you know.

best regards
Reinhard.



PD Dr. med. Reinhard von Wasielewski




------------------------------

Message: 2
Date: Mon, 19 Dec 2005 09:09:49 +1000
From: Laurie Reilly 
Subject: Re: [Histonet] CD4 and CD8 in mouse / human FFPE
To: wasielewski.reinhard.von@mh-hannover.de
Cc: histonet 
Message-ID: <5.1.0.14.0.20051219090417.00c4a238@mail.jcu.edu.au>
Content-Type: text/plain; charset="us-ascii"; format=flowed

Reinhard,
Could I please have a copy of your protocol, we have had lots of trouble 
trying to stain with
CD4 and CD8 in bovine tissues.
Any advice you can give would be most welcome, particularly about antigen 
retrieval.

      Hope everyone has a happy Christmas,
             Regards,
                         Laurie.

At 08:49 PM 18/12/2005 +0100, you wrote:
>Hi Patsy,
>we do a lot of CD4 and CD8 in human FFPE tissues, even in BM after 
>decalification.
>It works very good and is daily routine in our lab. If you need advice, 
>give me a mail.
>We now got some hints how both markers could work in mouse tissue, too. If we
>succeed, we'll let you know.
>
>best regards
>Reinhard.
>
>
>
>PD Dr. med. Reinhard von Wasielewski
>
>
>_______________________________________________
>Histonet mailing list
>Histonet@lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Mr.Laurie Reilly                                              Ph 07 4781 4468
School of Veterinary & Biomedical Science      Fax  07 4779  1526
James Cook University
Townsville  Qld. 
4811                                      laurie.reilly@jcu.edu.au 

Australia.




------------------------------

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End of Histonet Digest, Vol 25, Issue 24
****************************************

PD Dr. med. Reinhard von Wasielewski


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