After 24 h of treatment with the PM2.5, the cells were processed for immunofluorescence assay according to the standard protocol. Briefly, following three times of rinse with PBS, the cells were fixed with 4% paraformaldehyde (pH 7.4) for 30 min and washed three times with PBS. Then, the cells were permeabilized with 0.3% Trition X-100 at room temperature for 30 min and rinsed three times with PBS. Subsequently, following 1 h of blockage with 3% BSA in PBS, the cells were probed with the corresponding rabbit primary antibodies (anti-LC3 or anti-p62, 1:200 dilution) (Proteintech Inc., USA) in PBS containing 1% BSA overnight at 4 °C. Then, following three times of washing with PBS, the cells were labeled with a 1:500 dilution of fluorescein-marked second antibody (Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L)) for 1 h at room temperature protected from light. After three times of washing, nuclei were stained with DAPI (Beyotime Biotechnology, China) at 1:5000 dilution in PBS for 5 min in darkness. Subsequently, dishes were mounted with Prolong Gold Antifade Reagent (ThermoFisher Scientific Inc., Beijing), and fluorescent images were taken using the Nikon fluorescence microscope.
