High magnification fluorescence microscope

First, cells were seeded on coverslips in 24-well plates and were allowed to attach by overnight incubation. GFP-LC3 or GFP-RFP-LC3 vectors were transfected in HCT116, SW480 and HT29 cells, respectively, for 24 h, following treatment with DMSO (control), or BA for 48 h. After depletion of the medium, cells were washed with PBS three times, fixed by 4% paraformaldehyde for 15 min, washed with PBS three times and photographed with a high-magnification fluorescence microscope (Olympus Optical Co.).

Cells were seeded on coverslips in 24-well plates and were allowed to attach by overnight incubation. Following treatment with DMSO (control) or BA at indicated concentrations, cells were stained with 1 μg/ml acridine orange in PBS for 15 min, washed with PBS and examined under high-magnification fluorescence microscope (Olympus Optical Co., Hamburg, Germany).

Cells were seeded on coverslips in 24-well plates and were allowed to attach by overnight incubation following treatment with DMSO (control) or BA at indicated concentrations for 48 h. Cells were fixed in 4% paraformaldehyde at 4 °C for 15 min, permeabilized with 0.01% Triton X-100 for 5 min and then blocked with 10% BSA for 1 h. Cells were incubated with primary Ab (rabbit anti-human LC3 or/and LAMP1, diluted 1 : 200, respectively) overnight, and then incubated with secondary Ab (Alexa fluor 594 goat anti-rabbit or/and Alexa fluor 488 goat anti-mouse) for 1 h. Unbound Ab was removed with PBS; thereafter, cell nuclei were stained with DAPI (1 : 20). The samples were examined under a high-magnification fluorescence microscope (Olympus Optical Co.).