Automated immunostainer

The tissue blocks were cut into 4-μm sections and processed for immunohistochemistry. The lineage of EBV+ cells was determined by staining with antibodies against CD3, CD5, CD4, CD8, CD56, CD30, TIA1, and Ki67. Immunohistochemical stains were performed with an automated immunostainer (Ventana Medical Systems, Tucson, AZ), according to the manufacturer’s protocol. EBV was detected with the EBV Probe In Situ Hybridization Kit (TRIPLEX INTERNATIONAL BIOSCIENCES, CHINA, CO. LTD).

Immunohistochemistry on murine tissues was performed on an automated immunostainer (Ventana Medical Systems Inc., Oro Valley, AZ) according to the company’s protocols for open procedures with slight modifications. Appropriate positive and negative controls were used to confirm the adequacy of the staining. The histologic samples were analyzed by an experienced pathologist (L. Quintanilla-Martinez). Photomicrographic images were acquired with an Axioskop 2 plus Zeiss microscope equipped with a Jenoptik (Laser Optik System, Jena, Germany) ProgRes C10 plus camera and software. Objectives Plan-Neofluar used were as follows: 1.25/0,035, 2.5×/0.075, 10×/0.30, 20×/0.50, and 40×/0.75. Final image preparation was performed with Adobe Photoshop CS6 (Adobe Inc., San José, CA).

Formalin-fixed, paraffin-embedded tissues were sectioned at a thickness of 4 μm and stained using an automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA) according to the manufacturer’s protocol. The slides were dried at 60 °C for 1 h and deparaffinized at 75 °C for 4 min using EZ Prep (Ventana Medical Systems, Tucson, AZ, USA). The cells were conditioned (heat pre-treatment) at 100 °C for 64 min using a cell conditioning solution that contained Tris/borate/ethylenediaminetetraacetic acid. The anti-PD-L1 22C3 mouse monoclonal primary antibody (Agilent Technologies, Santa Clara, CA, USA) was diluted to 1:50 and applied to the sections, which were then incubated at 37 °C for 32 min. Signals were detected using an Optiview detection kit (Ventana Medical Systems, Tucson, AZ, USA) with streptavidin-biotin staining. Counterstaining was performed for 2 min at room temperature using Mayer’s hematoxylin (ScyTek, Logan, UT, USA). PD-L1 expression was defined if membranous and/or cytoplasmic staining was observed in tumor cells and tumor-associated immune cells. The combined positive score (CPS) was recorded based on the number of PD-L1-positive tumors and immune cells in relation to the total number of tumor cells. PD-L1 positivity was defined as a CPS > 1.