True nuclear transcription factor buffer set

For Ki67, DAPI, RELMα, BrdU and pHH3 staining, the eBioscience FoxP3/Transcription Factor Staining Buffer set (00-5523-00) or the BioLegend True-Nuclear Transcription Factor Buffer set (424401) was used. In brief, after surface staining, cells were fixed with 1x Fix Concentrate buffer in provided Fix Diluent for 30 minutes at 4 °C. Cells were then washed with FACS buffer and stored overnight. To permeabilize the cells, samples were washed with 1x Perm buffer diluted in water. Following blocking with 2% rat serum, samples were stained for 1 hour at RT, except for DAPI and secondary antibodies (20 minutes at RT), followed by washing in 1x Perm buffer and FACS buffer before flow cytometry.
For BrdU staining, mice were given 1 mg of BrdU i.p. (from BD kit, 552598, or Sigma, B5002) three hours before sacrifice as described^{12 (link)}. After sacrifice of mice and peritoneal lavage, samples were processed, fixed and stored overnight as for other intracellular antigens. BrdU-labelled cells were washed in 1x Perm buffer, incubated in DNase I (from BD kit, 552598 or Sigma, D4513) in 1x DPBS for 30 min at 37 °C, washed in 1x Perm buffer, blocked with 2% rat serum and stained for 1 hour at RT with α-BrdU antibody (BD, 552598), followed by washing in 1x Perm buffer and FACS buffer. Mice that did not receive BrdU were used as negative controls.