Rabbit anti pd 1

Staining for two groups of combination of either CD8/CD103 or CD8/PD-1 was performed on formalin-fixed paraffin-embedded (FFPE) lung tissue slides. FFPE slides were deparaffinized in CitriSolv for 30 min and then immersed in alcohol series from 100, 95, 85, and 75% to distill H₂O for 5 min each for tissue hydration. For antigen retrieval, hydrated slides were steamed for 20 min in 1 mM EDTA. The slides were then blocked with 10% normal goat serum phosphate-buffered saline (PBS) for 30 min at room temperature (RT) and then were incubated with either rabbit anti-CD103 (Abcam) or rabbit anti–PD-1 (Cell Signaling) overnight at 4°C.8After rinsing in 0.1% PBST (PBS with Tween 20) solution, the slides were incubated with Alexa Fluor 488–conjugated goat anti-rabbit secondary Ab (Life Technologies). After rinsing with 0.1% PBST, the slides were then incubated with Alexa Fluor 647–conjugated mouse anti- CD8 (BioLegend) for 60 min at RT. After stringent washing in 0.1% PBST, slides were aired before mounting with 4’,6-diamidino-2- phenylindole for nuclei counterstain. Tissue staining for the Ab mixture was reviewed and representative images were captured in Olympus cellSens Dimension system. Fifteen representative image fields were captured for each patient for quantification purposes.