Detecting transcription factor Foxp3 (mouse) easily

Results Summary

Freshly isolated cells were subjected to regular surface staining, followed by nuclear staining for Foxp3 as follows. FJK-16s clone is the most sensitive and stable in our hands. It works very well with the Foxp3 staining buffer kit from eBio (00-5523-00). We have reduced the use of the buffer volume and treatment time to make the nuclear staining process quicker: Fixation with 1:3 mixture of the concentrate and diluent in ~ 200 micro-liter at RT for 15 mins (spin plates at 1800rcf, 3 mins, invert plate and tap to completely remove fixative), followed by Foxp3 staining in ~40 micro-liter 1X permeabilization buffer at RT for 30 mins (then add ~300 micro-liter 1X permeabilization buffer, and spin plates at 2000rcf, 3mins, invert plate to remove washing buffer). Cells resuspended in 1X permeabilization buffer are ready for flow detection. Many surface staining antibodies are stable and work in Foxp3 staining buffer condition as well, and so the whole staining can be shortened into just the fixation and nuclear staining (with Fc blocking) as above. We recommend a quick test on all surface staining antibodies in your case before starting a large scale experiment.