2.5. BODIPY (493/503) dye

BODIPY (Life Technologies, Eugene, OR) assays were conducted to confirm differences in intramyocellular lipid content (mainly lipid droplets) and to correlate with PEX19 and palmitic and lignocerate oxidation. The staining and quantification procedure was based on previous publications [23-25]. Briefly, frozen human skeletal muscle was sectioned at 10 μm using a cryostat, mounted on slides (two cryosections per slide), rapidly fixed in ice-cold 4% paraformaldehyde solution for 1 h, and then rinsed with phosphate buffered solution (PBS) for 3 × 5 min. The sections were immersed in 10 μg/ml BODIPY working solution using Coplin jars for 30 min. Subsequently, the sections were rinsed in PBS 5 × 5 min, covered with coverslips using Vectashield mounting medium (Burlingame, CA) and sealed with transparent nail polish. Fluorescent images were captured with an optical microscope (Leica DM6000, Germany) through a 20× objective. A laser was used to excite BODIPY 493/503. Imaging settings were kept constant for all slides. Quantification of lipid droplets was performed using multi measurement plugin of ImageJ with a constant threshold to compare the fluorescent signal between subjects. Individual muscle fibers were manually delineated and measured.