Anti cd8 clone epr20305

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).