Comparative reactivity of “in house” hybridoma supernatants containing our FOXP3 monoclonal antibodies. Panel A illustrates immunoperoxidase labeling of the same area from sequentially cut sections from a single, paraffin-fixed and formalin-embedded tonsil, using previously described methodology.⁴  Tonsil tissue was collected with informed patient consent under ethical approval from the Oxfordshire Clinical Research Ethics Committee in accordance with the Declaration of Helsinki. Cells were counterstained with hematoxylin Gill No. 2 (Sigma-Aldrich, St Louis, MO), mounted in Aquatex (VWR International, Poole, United Kingdom) and viewed using a 40×/0.65 numerical aperture objective (Zeiss) on a bright field Axioskop microscope (Zeiss, Welwyn Garden City, United Kingdom). Images were captured using a Micropublisher 5MP RTV camera (QImaging, Surrey, BC). In terms of both frequency and intensity of FOXP3⁺ cell labeling, antibody 259D/C7 was as effective as the other clones. Panel B illustrates the full-length and truncated recombinant FOXP3 proteins that were expressed in COS-1 cells and their reactivity with the panel of FOXP3 antibodies by immunoperoxidase labeling. The expression of each truncated FOXP3 protein was also verified by detection of the Xpress epitope tag. The clone names in colored fonts indicate those antibodies with an epitope in the region of FOXP3 indicated by underlining in the same color. Numbers above the full-length protein give amino acid coordinates for FOXP3 truncations. Panel C shows Western blotting detection of FOXP3 expression in a lysate prepared from CD4⁺CD25⁺ peripheral blood mononuclear cells that were purified using a Regulatory T-Cell Isolation Kit (Miltenyi Biotech, Bisley, United Kingdom) from a blood buffy coat preparation obtained from the National Blood Service (Bristol, United Kingdom). Antibodies 259D/C7 and 206D/B1 strongly labeled both FOXP3 isoforms, while weaker labeling was observed with 236A/E7. As predicted, the smaller FOXP3 isoform lacking exon 2 was not detected by 150D/A3.