D7u8c 82723

To identify PD-1, mouse monoclonal antibody (mAb) NAT105/ab52587 (Abcam, Cambridge, UK) was used, citrate buffer was used to retrieve the antigen and positive signals were envisioned with MACH3 mouse HRP reagents (brown) (Biocare Medical, Walnut Creek, CA, USA). Rabbit mAb E1L3N/13684 (Cell Signaling Technology, Danvers, MA, USA) was used to identify PD-L1 and rabbit mAb D7U8C/82723 (Cell Signaling Technology) was used to identify PD-L2. As described in previous studies, E1L3N/13684 [34 (link)] and D7U8C/82723 [35 (link)] detect membranous expression of tumor cells and immune cells, respectively. In order to accurately distinguish between PD-ligands expressed on tumor cells or TILs, double immunostaining for PD-ligands and the B-cell biomarker PAX-5 was performed. Mouse mAb M7307/DAK-Pax5 (Dako, Santa Clara, CA, USA) was used to identify PAX-5. Antigens for PD-L1, PD-L2 and PAX-5 were retrieved in Tris-EDTA buffer (pH 9.0) in a pressure cooker. Positive signals for PD-L1 and PD-L2 were envisioned with the Betazoid DAB detection kit (brown) (Biocare Medical), and PAX-5 was detected using Warp Red chromogen (red) (Biocare Medical). Lastly, the sections were counterstained with Intellipath FLX hematoxylin. All antibodies were diluted 1:50.

Four µm FFPE sections were used for immunohistochemical (IHC) stainings. For IDO1, the Autostainer 480 instrument (Thermo Fischer Scientific, Waltham, MA) was used, and for PD-L2, the Intellipath FLX system (Biocare Medical, Pacheco, CA) was used. Rabbit polyclonal antibody HPA023149 (Atlas Antibodies AB, Stockholm, Sweden) (dilution 1:200) was used for IDO1, and rabbit monoclonal antibody D7U8C/82723 (Cell Signaling Technology, Danvers, MA) (dilution 1:50) was used for PD-L2. Antigen retrieval was performed using citrate buffer and TE buffer for IDO1 and PD-L2, respectively. The DAB Qaunto detection kit (Thermo Fischer Scientific, Waltham, MA) and the Betazoid DAB detection kit (Biocare Medical, Walnut Creek, CA) were used to envision IDO1 and PD-L2, respectively. Hematoxylin was utilized to counterstain the slides. Further details regarding IDO1 and PD-L2 stainings have been described previously [23 (link)]. EBV-encoded small RNA (EBER) in situ hybridization and IHC stainings for PD-1 and PD-L1 have been previously described in detail [22 (link)].