Fluorescent Immunostaining of PD-L1 Proteins

Cells were fixed in 4% PFA for 15 min, permeabilized with 0.5% Triton X-100 for 15 min and blocked with 10% goat serum for 1hr. For frozen tumor tissues staining, 40 μm sections were pre-treated with acetone for 10 minutes. After washing with PBS, cells were stained with rat antibody against mouse PD-L1 (clone 5C5, generated in the laboratory of Dr. Gordon Freeman^{52 (link)}), mouse antibody against human PD-L1 (clone 9A11, generated in the laboratory of Dr. Gordon Freeman^{50 (link)}) or anti-HA tag antibody for HA-ins-PD-L1 (#2367, Cell Signaling Technology). For HA staining, cells were incubated with Tyramide Boost Kit (#B40941, Thermo Fisher Scientific) according to the manufacturer’s protocol (tyramide incubation 15 min). Then cells were incubated with NucRed Dead nuclear stain (#R37113, Thermo Fisher Scientific) for 15 min and washed with PBS. For mouse PD-L1 or human PD-L1 study, cells or tissues were stained with a donkey anti-rat or donkey anti-mouse secondary antibody (Alexa Flour 488) for 1 hr. Nuclei were stained with DAPI. For Halo-tag imaging^{53 (link)}, cells were treated with 0.5 μM HaloTag Alexa Fluor 488 Ligand (#G1002, Promega) for 15 min. Images were captured using confocal microscopy (Nikon Ti inverted or Zeiss LSM880) and assembled using Fiji software.