Photometrics coolsnap ccd camera

For immunofluorescence, whole cells were washed in PBS, then spread on poly-lysine–coated slides. For cytoskeleton preparations, cells were first extracted with 0.25% NP40 in PIPES buffer (100 mM PIPES [pH 6.9], 1 mM MgCl2) for 5 min, and then washed twice in PIPES buffer. Cytoskeletons were fixed overnight in cold methanol at -20°C. For whole cells, slides were fixed for 5 min in 3% paraformaldehyde and neutralised for 10 min in 100mM Glycine, then permeabilized in 0.2% Triton X100 for 10 min. After two washes in PBS, whole cells and cytoskeletons were blocked in 1% bovine serum albumin (BSA) for 30 min. Cells were probed with anti-PFR2 (2E10B7) 1:2000, anti-HA (Sigma) 1:200, anti-mCherry (Gene Tex) 1:1000, anti-YL1/2 (Millipore) 1:1500 and anti-mNeonGreen (Chromotek) 1:1000 in 1% BSA overnight at 4°C. Slides were washed in 1% BSA before incubation with anti-IgG specific secondary antibodies conjugated to Alexa Fluor 488 (1:4000) or Alexa Fluor 546 (1:2000) (Invitrogen) for 1 h. Slides were washed in PBS, and DNA was stained with DAPI and mounted with Slowfade (Molecular Probes). Images were acquired using MetaView (Universal Imaging) software, on a Zeiss Axioplan 2 microscope equipped with a Photometrics CoolSnap CCD camera (Roper Scientific). Flagellum length measurements and processing of images were completed in ImageJ (https://imagej.nih.gov/ij/).

After 48-h DEX and/or r-irisin treatment, myotubes were fixed with 4% paraformaldehyde in phosphate-buffered saline. Images were from a Nikon Eclipse TE2000U microscope (Nikon, Avon, MA), captured using Photometrics Cool SNAP CCD camera (Roper Scientific, Tucson, AZ, USA) under phase-contrast microscopy at × 100 magnification. Diameters of individual myotubes were analyzed using MetaMorph 6.1 software (Molecular Devices, Sunnyvale, CA, USA). Average diameters of at least 200 myotubes were determined for each condition at three points separated by 50 μm along the myotube.