Ab130039

For mice, tumor sections and commercial antibodies (Abcam, Shanghai, China, ab130039) were used for IHC staining. For patients, tumor tissue samples obtained via core needle biopsy samples obtained from primary lesions were used for IHC staining. Rabbit monoclonal antibodies against PD-L1 (28-8, Abcam, Shanghai, China, AB205921) and PD-1 (EPR4877 (2 (link)), Abcam, Shanghai, China, ab137132) were used for IHC analysis. The reaction was visualized using SignalStain Boost IHC Detection Reagent (HRP, Rabbit). Tumor expression of PD-1 or PD-L1 was considered positive when membrane staining was observed. The following semiquantitative scoring method (6-step scoring system, ‘Cologne Score’) was used for PD-1 and PD-L1: 0 (<1%), 1 (≥1% and <5%), 2 (≥5% and <10%), 3 (≥10% and <25%), 4 (≥25% and <50%), 5 (≥50% and <75%) (17 (link), 18 (link)). Tumors with a score ≥2 were graded as relatively high expression, and tumors with a score ≤1 were graded as having relatively low expression (a cut-off value of 5%). An Eclipse Ci-L photographic microscope (Nikon, Japan) and Media Cybemetics software (USA) were used to analyze tumoral expression of PD-1 and PD-L1. Two experienced pathologists were consulted to ensure the accuracy of semi-quantitative scoring approach used.