PD-L1 Immunohistochemistry in HNSCC

The University of Pittsburgh IRB #99–069 approved the use of clinical samples, and written informed consent was obtained. Slides were deparaffinized and rehydrated. Antigen retrieval was performed using Diva Retrieval (Biocare Medical) and a decloaking chamber at 124°C, 3 minutes, and cooled for 10 minutes. Slides were placed on an Autostainer Plus (Dako) using a TBST rinse buffer (Dako) and stained using 3% H₂O₂ (Thermo Fisher Scientific) for 5 minutes, CAS Block (Invitrogen) for 10 minutes, and the primary antibody for PD-L1 (6.2 μg/mL final concentration, clone 405.9A11, kindly provided by Gordon J. Freeman) was used as previously reported (32 (link)). The secondary consisted of Envision Dual Link + (Dako) polymer for 30 minutes, rinsed, then a TBST holding rinse was applied for 5 minutes. The substrate used was 3,3,-diaminobenzidine + (Dako) for 7 minutes and counterstained with hematoxylin. PD-L1 staining was quantified by positive pixel count v9 algorithm (Aperio). A head and neck pathologist blinded to clinical patient data examined tumor sections. Scoring was determined by the percentage of tumor stained for PD-L1. Tumors with <5% tumor cell-positive staining were considered negative.