1.4 na oil immersion objective

Cells were cultured on coverslips for 24 hours, washed trice with Phosphate-buffered saline (PBS), and then fixed using 4% paraformaldehyde in PBS at room temperature (RT) for 10 minutes followed by 3 washes in PBS. Cells were incubated for 3 minutes at RT in 1 M Glycine solution to avoid quenching followed by 3 washes in PBS. Cells were permeabilized using 0.1% of Triton-100X in PBS for 10 min at RT and washed trice with PBS. To block not-specific binding sites, cells were incubated at RT for 45 min in PBS with 4% FBS and 1% BSA (blocking buffer). Cells were incubated with primary antibody diluted in blocking buffer for 1 hr at RT. Cells were washed with PBS and incubated with secondary antibody diluted in blocking buffer for 1 hr at RT followed by 3 washes in PBS. Coverslips were dried and then mounted on slide using Fluoromount-G mounting media.
Immunofluorescent samples were imaged using a laser scanning confocal microscope LSM 710 NLO (Zeiss, Jena, Germany) equipped with 63×/1.4NA oil-immersion objective (Zeiss). An Argon 488 laser 40 mW (Green), DPSS laser 561 20 mW (Red), and Helium-Neon 633 (Far red) were used to excite Alexa Fluor 488, Alexa Fluor 561, and iRFP fluorophores respectively. Samples were visualized on standard photomultiplier tube (PMT) detector.

OCT embedded sections were fixed with acetone for 15 min at room temperature and stained with anti-Tomm20 antibody conjugated with a 647 nm excitation fluorophore (Abcam, ab209606). A Zeiss LSM 880 confocal microscope in Airyscan mode was used to visualize mitochondria in liver sections, exciting the sample with a 633 nm laser and imaging the sections with a Zeiss 63 × /1.4NA oil immersion objective. 20 fields of images containing 3–6 cells per liver section were collected for 6 mice per experimental condition. Mitochondria within areas of interest were individualized through the CellProfiler cell image analysis software (https://cellprofiler.org/). To minimize background, images were subjected to a median filter. Segmentation of mitochondria was performed by utilizing a global, two-class, otsu-thresholding method and minimizing the weighted-variance to shape. Identified objects were then subjected to automated shape descriptor analysis, which was used to measure the area of each individual mitochondrion. Data are presented as symbols that represent the average individual mitochondrion area per imaged field. Representative images shown were adjusted in brightness and contrast for better visualization.

Based upon the results from live cell imaging, we picked the 48‐h time point for detailed examination in fixed cells. Human primary myotubes from three different donors were grown on #1 coverslips and incubated with resveratrol for 48 h. Myotubes were fixed with 3.7% formaldehyde and stained with 286 nM DAPI (4’,6‐diamidino‐2‐phenylindole, dihydrochloride; D1306; Invitrogen) to visualize the nuclei and stained with Bodipy 493/503 (1:100) to visualize the LDs. Coverslips were mounted on glass slides with Mowiol. Imaging was performed with a FEI Corrsight spinning‐disk confocal microscope, using a 63× 1.4 N.A. oil immersion objective (Zeiss) and with a Nikon E800 fluorescence microscope (Nikon), coupled to a Nikon DS‐Fi1c colour CCD camera (Nikon), using a 40x objective. Images obtained from the Corrsight were analysed similar to the live cell images. Images from the Nikon microscope were analysed upon thresholding and Bodipy‐derived signal was corrected for the number of nuclei.