Fluoroshield with dapi

C2C12 myoblasts stably transfected with Ad-GFP-LC3 (Hanbio, Shanghai, China) were seeded on glass cover slips. Twenty-four hours after transfection, the cells were treated with or without APN (30 μg/mL up to 24 h). The cells were then stimulated with H₂O₂ for 30 min. After that, cells were fixed for 15 minutes with 4% paraformaldehyde and washed twice with PBS 1X. Cells were blocked and permeabilized with PBS 1X + 0.2% Triton X-100 for 15 minutes at room temperature. After washing twice with PBS 1X, cells were incubated with a rabbit monoclonal TOMM20 antibody (Abcam) diluted 1:250 in 5% BSA O/N at 4 °C and washed twice with PBS 1X followed by incubation with a secondary anti-rabbit IgG antibody, conjugated to Alexa 555 (1:200) for 1 hour at room temperature. Coverslips were washed twice with PBS 1X and mounted on glass slides with fluorescent mounting medium Fluoroshield™ with DAPI (eBioscience) and visualized in an Olympus FV1000 confocal microscope.
To determine colocalization between mitochondria and lysosomes, C2C12 myoblasts were loaded with 200 nM MitoTracker Green FM (Beyotime, C1048) and 50 nM LysoTracker Red (Beyotime, C1046) for 30 min at 37 °C. Cell images were obtained using an Olympus FV1000 confocal microscope. The colocalization of mitochondria and lysosomes was analyzed according to the number of mitochondria-localized lysosomes.

C2C12 myoblasts stably transfected with Ad-mRFP-GFP-LC3 (Hanbio, Shanghai, China) were seeded on glass cover slips. Twenty-four hours after transfection, the cells were treated with or without APN (30 mg/mL up to 24 h). The cells were then stimulated with H₂O₂ for 30 min to induce autophagy. Coverslips were washed twice with PBS 1X and mounted on glass slides with fluorescent mounting medium Fluoroshield™ with DAPI (eBioscience) and visualized in an Olympus FV1000 confocal microscope.

For immunofluorescent microscopy of GS-5 10,000 to 12,000 cells were seeded on Laminin-coated 8-well chamber slides (Falcon, Corning, Amsterdam, NY, USA). Laminin-coating (10 µg/mL, sigma, L2020) was performed at 4 °C overnight. One day after seeding the cells were treated as indicated and fixed with 4% paraformaldehyde for 20 min at RT. The slides were washed with TBS-Tween (0.1%; TBS-T), blocked with 4% BSA in TBS with 0.3% Triton X-100 for 1 h at RT and primary antibody incubation occurred at 4 °C overnight. Hereafter, the slides were washed at least three times with TBS-T and secondary antibody was diluted 1:500 in TBS-T and incubated for 1 h at RT. After an additional wash with TBS-T the slides were mounted with DAPI containing Immunoselect antifading mounting medium (Dianova, Hamburg, Germany) or Fluoroshield with DAPI (Thermo Fisher, Frankfurt, Germany). Images were acquired with an Eclipse TS100 inverted fluorescence microscope (Nikon, Düsseldorf, Germany) operated by NIS Elements AR software (version 3.22, Nikon) or an AxioImager Z1 (Carl Zeiss, Jena, Germany).