[Histonet] RE: CD4/CD25/TGFB Antibodies; Filter choices for Leica

[C.M. van der Loos]

Dear Juan,
Persumed your investigation is on human tissues I have the following suggestions for you.
For double staining of CD4/CD25 and CD8/CD25 I would strongly recommend you to focus on cryostat sections for two reasons. In the first place I have tested a couple of CD25 antibodies that suppose to work on FFPE sections but so far I am not very impressed. CD25 staining on cryo's seems much more reliable to me (DAKO, Becton&Dickinson, etc.). In the second place, the performance of fluorescence staining techniques on FFPE sections is not as ideal because of autofluorescence of formaldehyde-fixed tissues. Again, acetone-fixed cryostat sections do not share this type of problem.
For a protocol you may test this:

1. CD4 or CD8
2. GAM/fluorochrome 1
3. Normal Mouse Serum
4. CD25/fluorochrome 2

On the otherhand the IHC staining of TGFB on cryo's, should be considered as tricky and perhaps even impossible (despite many papers that have been published...). The point is that TGFB is a small protein that tends to leak away from your cryo's (see: JHC 49:699-709, 2001 "IHC detection of IFNgamma: fake or fact?"). Although I don't have personal experience with TGFB, I have heard about problems about the IHC detection completeley similar to our IFNgamma IHC visualization failure. TGFB is perhaps detected much more reliable using optimally fixed FFPE sections. Given of course that the antigen is surviving the embedding procedure.
Lots of success,
Chris van der Loos, PhD
Dept. of Pathology
Academical Medical Center
Amsterdam - The Netherlands  

----- Original Message ----- 
>From  Juan Solon <Juan.Solon <@t> lshtm.ac.uk> 
Date  Wed, 24 Mar 2004 09:36:30 +0000 
To  histonet <@t> lists.utsouthwestern.edu 
Subject  [Histonet] CD4/CD25/TGFB Antibodies; Filter choices for Leica 
Dear All,

As part of a study on mucosal immunity in The Gambia, W. Africa, I plan to double stain antral and duodenal specimens for CD4 and CD25 & CD8 and CD25.  Also, we would attempt to triple stain for TGF beta on these subsets.  We have access to a Leica DMRXE although the filter cubes are all currently long pass suppression filters . I have both formalin-fixed and frozen sections of the antrum and the duodenum.  

I would like to ask for advise on several points:

1. Can anyone share their experience on antibody combinations known to work for CD4-25, CD8-25 and triple staining for CD4-CD25-TGFB either with vectastain elite (peroxidase) or immunofluorescence.  I have compiled a list of CD4, CD8, CD25, TGFB antibodies from Dako, Ancell, Novocastra, BD, and Serotec, but before purchasing any of these, I thought it would be wise to ask advise from the Histonet community.

2. I would probably need to acquire different filter cubes to do simultaneous imaging of fluorescence.  There are several suppliers I have encountered on the net (Omega Filters and Chroma Technology) * does anyone have experience on using filters from these companies on Leica microscopes (specifically the DMRXE).  Any problems encountered with using third-party filters with Leica microscopes?

Thanks.
Juan


Dr. Juan Antonio Solon
MRC Keneba Field Station
MRC Laboratories Gambia
Atlantic Road, Fajara
PO Box 273, Banjul
The Gambia
Email :  juan.solon <@t> lshtm.ac.uk
Tel No:  (++220) 541021
Fax No: (++220) 541022 / (++220) 496513