I did it three times to stain CD4+ T cells from the spleen, and I found the foxp3 positive cells were 100%, but CD25+Foxp3+ cells were 6-10%. I use the Foxp3 staining kit to permeate the cells. I am so confused about it. I think there are some probable reasons:
1. I did not use mouse serum to block after permeating the cells
2. I use the Fix/Perm buffer at 4 degrees for 1 -2hours overnight.
But I am nor sure about that.
Qiuju Chen,
The specificity of the staining can totally depend on the clone you use for Foxp3 and thus how you may or may not need mouse serum block. I recommend a good clone from Cell Signaling Technology (FoxP3 (D6O8R) Rabbit mAb #12653). To achieve good staining, you can block your tissue first with mouse Fcblock in 1X BD Cytoperm/wash buffer for 30min, then stain with 1:50 dilution of Foxp3 antibody at 4C overnight or RT 2-3hrs. Then wash and add secondary Donkey-anti-rabbit antibody (I recommend thermofisher alexa fluor plus series) and incubate at RT for 1 hr.
I Hope this is helpful to you! Good luck with your staining.
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