Diva retrieval

Slides were deparaffinized and rehydrated. Antigen retrieval was performed using Diva Retrieval (Biocare Medical, Concord, CA) and a decloaking chamber at 124 °C, 3 min, and cooled for 10 min. Slides were placed on an Autostainer Plus (Dako, Carpenteria, CA) using a TBST rinse buffer (Dako) and stained using: 3% H₂O₂ (ThermoFisher Scientific, Pittsburgh, PA) for 5 min, CAS Block (Invitrogen, Grand Island, NY) for 10 min, the primary antibody for STING (ab92605) (33) used per instructions. The secondary consisted of Envision Dual Link + (Dako) polymer for 30 min, rinsed, then a TBST holding rinse was applied for 5 min. The substrate used was 3,3, Diaminobenzidine + (Dako) for 7 min and counterstained with hematoxylin. STING staining were quantified by positive pixel count v9 algorithm (Aperio). A head and neck pathologist blinded to clinical patient data examined tumor sections. H-score of STING expression was calculated [25 (link)]. STING protein was expressed as an H score, defined as STING intensity multiplied by the percentage of STING positive cells.

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, Concord, CA) and a decloaking chamber at 124°C, 3 minutes, and cooled for 10 min. Slides were placed on an Autostainer Plus (Dako, Carpenteria, CA) using a TBST rinse buffer (Dako) and stained using: 3% H2O2 (ThermoFisher Scientific, Pittsburgh, PA) for 5 minutes, CAS Block (Invitrogen, Grand Island, NY) for 10 minutes, the primary antibody for PD-L1 (clone 405.9A11) (33 (link)) and the pJAK2 (Y1007-1008) (clone E132) used per instructions. The secondary consisted of Envision Dual Link + (Dako) polymer for 30 min, rinsed, then a TBST holding rinse was applied for 5 min. The substrate used was 3,3, Diaminobenzidine + (Dako) for 7 minutes and counterstained with hematoxylin. PD-L1 and pJAK2 staining were 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 %tumor stained for PD-L1 or pJAK2, respectively. Tumors with <5% tumor cells positive staining were considered negative.