2.6. Double immunofluorescence staining

The cells were seeded into round coverslips and treated as indicated. After washing twice with PBS, the cells were fixed with 3% paraformaldehyde for 10 minutes at 37℃ and permeabilized with 0.1% BriJ 98 for 90 seconds at RT. The samples were blocked with 10% goat serum for 1 hour at RT and incubate with anti‐PD‐L1 primary antibodies overnight at 4℃. Next, the coverslips were washed with PBS three times and incubated with goat anti‐rabbit IgG‐Alexa Fluor® Plus 647 secondary antibody for 2 hours at RT. The samples were blocked again and incubated with anti‐Casp8 or anti‐A20 antibodies for 3 hours at RT, followed by goat anti‐mouse IgG‐Alexa Fluor® 488 secondary antibody after washing three times. Nuclear staining was performed by DAPI. A confocal microscope (N‐STORM & A1) was used for image analysis.