For in vitro experiments, after treatment, HepG2 cells were fixed using 4% paraformaldehyde in PBS for 20 min at room temperature, washed twice in PBS, and blocked for 45 min at room temperature in PBS containing 6% BSA and 0.25% Triton X-100. Cells were then stained with rabbit anti-LC3 antibody (1:200 dilution, Novus) and FITC-labeled secondary antibody (1:2000 dilution, KPL).
For in vivo experiments, paraffin-embedded tissue sections were treated with improved citrate antigen retrieval solution (Beyotime) for 30 min at 100 °C. Following twice wash with PBS, they were treated with 0.25% Triton X-100 for 15 min and blocked with PBS containing 6% BSA for 45 min at room temperature. Then, tissue sections were stained with rabbit anti-LC3 antibody (1:200 dilution, Novus) or rabbit Atg13 antibody (1:200 dilution, Cell Signaling, 13468S) and Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) or Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 594 Conjugate) secondary antibody (1:300 dilution, Cell Signaling, 4412S/8889S).
All stained samples were visualized under a laser confocal microscope (Leica TCS SP5).
For in vivo experiments, paraffin-embedded tissue sections were treated with improved citrate antigen retrieval solution (Beyotime) for 30 min at 100 °C. Following twice wash with PBS, they were treated with 0.25% Triton X-100 for 15 min and blocked with PBS containing 6% BSA for 45 min at room temperature. Then, tissue sections were stained with rabbit anti-LC3 antibody (1:200 dilution, Novus) or rabbit Atg13 antibody (1:200 dilution, Cell Signaling, 13468S) and Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 488 Conjugate) or Anti-rabbit IgG (H+L), F(ab’)2 Fragment (Alexa Fluor® 594 Conjugate) secondary antibody (1:300 dilution, Cell Signaling, 4412S/8889S).
All stained samples were visualized under a laser confocal microscope (Leica TCS SP5).