Delayed FoxP3 expression in CD4+CD8-CD25+/- cells in neonatal thymus. A: Generation and characterization of the anti-FoxP3 antibody.293T cells were transfected with either vector or pHSPG-mFoxp3. FoxP3 was specifically detected by Western blot with the anti-FoxP3 antibody (top left), immunofluorescence (IF) staining (lower left), by FACS analysis (middle). The anti-FoxP3 antibody specifically stained intracellular FoxP3 in CD4+CD8-CD25+ Treg cells from wild type, but not scurfy, mice (right). B: Expression of FoxP3 protein is not detected in CD4+CD8-CD25+ thymocytes until 3 days after birth. Percentages of CD4+CD8-CD25+ and CD4+CD8-CD25- thymocytes (gate on the CD4+CD8- population from Fig. 1A) that expressed FoxP3 are shown, with the control IgG as background.