[Histonet] CD25 on murine tissue woes

From:Gayle Callis


You wrote:
I am trying to get CD25 staining to work in order to identify T reg cells 
in mouse tissue sections. So far I really haven't had much luck.  I have 
both a 7D4 biotinylated anti-CD25 from Pharmingen and a PC61 
Fitc-conjugated anti-CD25 from eBioscience and I am using Spleen as a 
positive control.
I am using fresh frozen sections that have not been fixed at all.  I have 
tried various fixations with Acetone and PFA.  I would think PFA might 
block staining as many people have had trouble getting staining on parafin 
sections.  I have played with many different antibody concentrations and we 
have secondaries and amplification protocols that have worked for other 
biotinylated and fitc-conjugated antibodies quite well using strep-hrp and 
anti-fitc-hrp along with a TSA cy3 or fluorescein kit.  Does anyone have a 
protocol or some tips on making these antibodies stain for 
immunofluorescence?  I know this is a common stain, but I simply have had 
no luck with it.  Any help is appreciated thanks.

Could you provide more details on HOW you are doing the staining, both as 
IHC and or immunofluorescence.  If you are trying to do the primary 
antibody directly conjugated to FITC on a tissue section, you may not get 
positive results - this is not uncommon with some of this type conjugate as 
a direct IFA stain.  I can't make it work with CD4-FITC or CD8-FITC.

As for biotinylated antibodies (primaries) one can do either IHC and IFA - 
we do this on many other rat antimouse primaries, CD markers - so provide 
concentrations, times, etc, etc - all those good things.

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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