Goat sera

For CD1d tetramer immunofluorescence, fresh thymic lobes, whole lymph nodes or 1–2 mm thickness spleen trans-sections were overnight incubated in 50μl phosphate buffered saline (PBS) solution containing 1:10~1:25 dilution of R-Phycoerythrin (PE) labeled PBS-57 loaded CD1d tetramer in 2% fetal calf serum containing PBS at 4°C. Next day, tissues were washed three times with PBS, fixed with 4% paraformaldehyde (PFA) for one hour and snap frozen. Five micrometer tissue sections were blocked with 5% bovine serum albumin and goat sera (Jackson Laboratory) for 1 hour at 25°C and stained with anti-PE antibody (Novus Biologicals, rabbit polyclonal) followed by goat anti-rabbit AF555 (Life Technology). For human CD2 immunofluorescence, tyramide based amplification was done according to manufacturer’s instruction (PerkinElmer) after staining with anti-human CD2 (RPA2) antibody. RORγt (RORg2, Millipore or Q31-378, BD) and PLZF (D9, SantaCruz or R17-809, BD) were stained followed by goat anti-hamster IgG (Jackson Laboratory) and goat anti-mouse IgG1 (Southern Biotech).