Anti pd l1 clone 22c3

A 5-µm-thick cross-section of a paraffin-embedded block was moved to a silanized glass slide. The sections were then de-paraffinized using xylene and rehydrated using a series of graded alcohols. Antigen retrieval was performed using a microwave vacuum histoprocessor (RHS-1; Milestone, Bergamo, Italy) by heating samples in 0.01 M citrate buffer (pH 6.0) for 20 min to a final temperature of 121 °C. The section was incubated for 10 min with hydrogen peroxide (3%) in methanol to prevent endogenous peroxide activity. Slides were then incubated with anti-CD3 (Abcam), anti-CD68 (clone: KP1, Dako, Carpinteria, CA, USA), and anti-PD-L1 (clone: 22C3, Dako) antibodies. After washing, the EnVision+ system HRP-labelled polymer (Dako) was used at 24 °C for 5 min. The slides were treated with 3,3′-diaminobenzidine for 5 min and then counterstained with hematoxylin.

Morphology of patient tumor tissue and their corresponding PDX models was studied by an expert pathologist. Histopathology of primary tumors and PDX followed standard protocols for HNSCC staging including HE staining and immunohistochemistry; antibodies: anti-CD8 (Clone C8/144B, Dako, Hamburg, Germany, anti-p16 (clone: G175-405, BD Bioscience, Heidelberg, Germany), anti-Ki-67 (Clone Mib-1, Dako), anti-PD-L1 (Clone 22C3, Dako).

The morphology of the patients’ tumor tissue and their corresponding PDX was studied by an expert pathologist. Histopathology of primary tumors and PDX followed standard protocols for HNSCC diagnostics including HE staining and immunohistochemistry; antibodies: anti-p16 (clone: G175-405, BD Bioscience, Heidelberg, Germany), anti-Ki-67 (Clone Mib-1, Dako, Glostrup, Denmark), anti-PD-L1 (Clone 22C3, Dako), and anti-p53 (Clone Do7, Dako). Standard immunoperoxidase technique was applied using an automated immunostainer, Autostainer Link48 (DAKO) with diaminobenzidine as chromogen.