Immunofluorescent Staining for Autophagy

immunofluorescent staining was performed as described previously by us^{46 (link),47 (link)}. Briefly, 2 × 10³ cells were plated into chamber slides (Millipore), and then were incubated with primary antibody against LC3 or p-AKT overnight at 4 °C. After washing with 1× PBS for three times, cells were incubated with secondary antibodies conjugated with Alexa Fluor 594 for 1 h. The slides were washed three times with 1× PBS, counterstained with DAPI, mounted and stored at 4 °C under dark conditions. Autophagy was quantified by quantification of LC3 puncta per cell using Leica TSC SP8 confocal laser scanning microscope. The average number of LC3 puncta per cell was counted with at least 100 cells for each cell line. The images were taken under confocal laser scanning microscope (LC3) or Leica DMI4000 fluorescenc microscope (p-AKT).