Hi Everyone,

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.



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