Fluoview 1000 confocal laser scanning system

Cells were seeded directly on glass coverslips and after 48 h were fixed with 4% paraformaldehyde and permeabilized by incubation with 0.2% Triton X-100. For mitophagy analysis, C2C12 cells were co-transfected with scramble (scr) or PGC-1α siRNA (PGC-1α(−)) and EGFP-LC3 and incubated with mouse HSPA9 (1 : 50) antibody. After staining with the appropriate AlexaFluor-conjugated secondary antibodies (1 : 1000), cells were incubated with Hoechst 33342 (Life Technologies, Carlsbad, CA, USA) to visualize nuclei. Images were visualized with an Olympus Fluoview 1000 confocal laser scanning system (Segrate, Milano, Italy). The images presented were captured under constant exposure time, gain and offset. Pearson's correlation coefficient, R(r), was calculated for fluorescence intensities of both the confocal channels. This coefficient describes the correlation between the intensity distribution or pattern overlap in two channels in terms of a least squares fit. This value can be between −1 and 1, and R=1 indicates complete correlation between the two channels. Finally, the overlap coefficient indicates an overlap of the signals and thus represents the true degree of colocalization.

Immunofluorescence assays for DRG2 were performed on paraffin-embedded muscle, heart, and liver tissue sections. The sections were incubated in different concentrations of ethanol and washed in PBS. The sections were boiled in an antigen retrieval buffer (10 mM sodium citrate buffer, pH 6.0, 0.5 ml Tween 20) for 10 min and washed in PBS. The sections were incubated in 0.3% H₂O₂ for 10 min at RT and blocked with 5% Normal Goat Serum for 1.5 h at RT. The sections then were incubated with primary antibodies against DRG2 (1 : 250 dilution in 5% Normal Goat Serum) overnight at 4°C, followed by the fluorescence-labeled secondary antibody Alexa Fluor 568 goat anti-rabbit (1 : 1,000). Nuclei were stained with DAPI (1 : 2,000) for 5 min at RT. The sections were mounted on glass slides and viewed on an Olympus FluoView 1000 confocal laser scanning system.

For histological analysis, tissues were fixed in 10% formalin solution, embedded in paraffin, and sectioned. H&E staining were performed as described previously [22 ].
For immunofluorescence (IF) staining of tissue and primary adipocytes, snap-frozen tissues were sectioned using a cryostat and then defrosted at room temperature for 1 h. The primary adipocytes and tissue sections were fixed in 4% paraformaldehyde (HT5014; Sigma-Aldrich) at room temperature for 20 min, washed thrice in PBS, incubated with 0.2% Triton X-100 for 5 min, and rinsed in PBS. The sections were blocked with 5% goat serum with 0.1% Triton X-100, whereas adipocytes were blocked with 3% bovine serum albumin (BSA) for 1 h at room temperature. Then, both were incubated with the target protein antibody (1:200) with their respective blocking solution overnight at 4 °C. The next day, the tissues and adipocytes were again washed thrice with PBS, stained with Alexa Fluor-conjugated secondary antibody in blocking solution at room temperature in the dark for 1 h, washed twice with PBS, and incubated with 10 μM DAPI in PBS at room temperature for 10 min. Images were acquired and analyzed using an Olympus FluoView 1000 confocal laser scanning system. The slides were scored in a blinded manner.