Pe conjugated secondary antibody

Flow cytometry was performed as previously described.^{24 (link)} in brief, HN4 and HN30 cells were incubated with the indicated agent for 48 h. The cells were collected and incubated with anti-human PDL1 antibody at 1:100 (BD Biosciences, Franklin Lakes, NJ) for 30 min on ice. Then, the cells were resuspended in 100 μl fluorescence-activated cell sorting buffer and analysed on BD Fortessa flow cytometer. The final results were analysed with FlowJo software. Signal intensity was calculated as the ratio of the median fluorescence of the PDL1 antibody to that of the isotype control antibody (SFI: specific fluorescence index). CD4-FITC antibody, CD8-PerCP-Cy5.5 antibody, CD56-APC antibody, and PD1-PE antibody (all purchased from BD Biosciences) were applied to detect the PD1 expression on the surface of immune cells from peripheral blood of HNSCC patients and healthy controls. The IFNAR1 antibody (Abcam, Cambridge, MA, UK) and PE-conjugated secondary antibody (Proteintech, Rosemont, IL, USA) were used to analyse the surface IFNAR1 expression on immune cells.

The deparaffinized slides were boiled in citrate buffer at 100°C for one hour for antigen retrieval and then were blocked in 1% FBS at room temperature followed by overnight incubation with primary antibodies. The primary antibodies, including CD31, α-SMA, and CD68 (CST, USA), were used to incubated with the sections, respectively, followed by incubated with PE-conjugated secondary antibody (Proteintech, Wuhan). Finally, cell nuclei were stained with DAPI. Images were captured and processed using a fluorescence microscope (IX 53, Olympus Corporation, Japan).