Immunofluorescence staining of LC3

H9C2 cells from each group were washed with PBS three times and fixed with 4% paraformaldehyde for 15 min at 4°C. Cells were permeabilized with Tris-buffered saline (TBS) containing 0.25% Triton X-100 (TBSX) three times for 10 min each at 4°C, and then blocked with 10% horse serum (cat. no. H8890; Sigma-Aldrich; Merck KGaA) in TBSX for 1 h at 4°C. Subsequently, cells were incubated with a primary antibody against LC3 (1: 500; cat. no. NB100-2220; Novus Biologicals Canada ULC, Oakville, ON, Canada) overnight at 4°C, followed by incubation with a swine anti-rabbit FITC-conjugated secondary antibody (1:50; cat. no. F0205; Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) for 30 min at 4°C. Cells were then stained with 1 µg/ml DAPI at 37°C for 5 min. Cells were mounted with anti-fading medium. Three randomly chosen microscopic fields were analyzed under a confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) and quanitified using ImageJ software version 1.48 (National Institutes of Health).