Tcs sp8 confocal laser scanning microscopy platform

To detect the cellular uptake of PA-ELNs, dilinoleyl Dil with orange‒red fluorescence was employed to label PA-ELNs. Cells were incubated with dilinoleyl Dil-labeled PA-ELNs for 3 h. The treated cells were fixed in 4% paraformaldehyde for 10 min. The nuclei were labeled with DAPI (blue fluorescence), while cytoskeleton were labeled with phalloidin (green fluorescence). Meanwhile, anti-fluorescence quenching seal solution was added to slow fluorescence quenching. Finally, the cells fluorescent images were visualized by a TCS SP8 confocal laser scanning microscopy platform (Leica, Germany) [27 , 28 ].

HepG2 cells were seeded onto glass coverslips in a 12-well plate and treated with vehicle or AA for 24 h. After treatment, cells were fixed with 4% paraformaldehyde (PFA) for 10 min then rinsed in PBS three times for five minutes. Cells were blocked in antibody dilution buffer containing 5% FBS and 0.3% Triton X-100 in PBS for 1 h at room temperature. After blocking, cells were incubated with anti-LC3B (1:1000 dilution; GeneTex) overnight at 4 °C. Cells were then rinsed with PBS three times for five minutes and blotted with Alexa Fluor^TM 488-conjugated anti-rabbit secondary antibody for 1 h at room temperature. Cells were rinsed in PBS three times for five minutes before mounting in ProLong Diamond Antifade mounting medium with DAPI. Cells were imaged on a Leica TCS SP8 confocal laser scanning microscopy platform (Wetzlar, Germany). The LC3 puncta in each cell were calculated by a researcher who was blinded to the sample identity.