Pd l1 clone 28 8

FFPE tumor tissue was used for single chromogenic IHC analysis of PD-L1 (clone 28-8, dilution 1:100; Abcam) in malignant cells. The antibody’s conditions were previously optimized and validated⁴¹ . The IHC staining was performed in a Leica Bond Max autostainer system (Leica Biosystems) according to standard automated protocols. Briefly, tissue sections were deparaffinized and rehydrated following the Leica Bond protocol; antigen retrieval was performed with Bond Solution no. 2 (Leica Biosystems, equivalent to ethylenediaminetetraacetic acid, pH 9.0) for 20 min; the primary antibody was incubated for 15 min at room temperature and detected using the Bond Polymer Refine Detection kit (Leica Biosystems) with 3,3′-diaminobenzidine as the chromogen. The slides were counterstained with hematoxylin, dehydrated and cover-slipped. Two pathologists evaluated PD-L1 expression in the membrane of viable malignant cells by standard microscopy according to the International Association for the Study of Lung Cancer guidelines⁴² . The results were reported as percentage of malignant cells with any positive membrane staining. Experiments and scorings related to the presented micrographs were conducted once. The results were plotted using Microsoft Excel v.2016 and GraphPad Prism v.8.00.