To evaluate the binding antibody to intratumoral Tregs, multiplex immunohistochemistry (IHC) for tumor infiltrating lymphocytes (TILs) in tissue sections was carried out using Opal 7-Color Automation IHC Kit (Akoya Biosciences, Hopkinton, MA) and BOND RXm auto stainer (Leica Biosystems, Wetzlar, Germany). Anti-CTLA4-IgG (1 mg, 6.8 nmol) or antiCTLA4-F(ab′)2 (0.73 mg, 6.8 nmol) antibody was conjugated with a 5-fold molar excess of digoxigenin (DIG) NHS ester (MilliporeSigma) in Na2HPO4 (pH 8.5) at room temperature for 1 h. The mixture was purified with a Sephadex G25 column (PD-10). Each DIG-conjugated antibody was injected into MC38-luc tumor-bearing mouse via tail vein. The tumors were extracted 24 h after injection, then fixed with 10% formalin, embedded in paraffin, and thinly sliced. The following antibodies and DAPI were used: anti-CD8 (clone EPR20305; Abcam, 1:500 dilution), anti-Foxp3 (clone 1054C; Novus Biologicals, 1:1,000 dilution), and anti-DIG (clone 9H27L19; Thermo Fisher Scientific, 1:500 dilution) antibodies. The staining using this system was carried out as previously described.25 (link) Stained slides were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were captured by Mantra Quantitative Pathology Workstation (AKOYA Biosystems) and were analyzed with inForm software (AKOYA Biosystems).