Fm1 43

WT (C25) and DYSF-null (AB320) human myoblasts were plated at 50% confluence on a 6-well plate (VWR) and transfected using Lipofectamine 2000 (Invitrogen) per manufacturer’s directions. After 24 h, cells were visualized in the presence of Ca2+ (1 mM) and membrane-impermeable dye FM 1-43 (2.5 µM, Molecular Probes) with a confocal microscope (LSM ×800, ×63 objective, ZEISS). Membrane damage was applied to myoblasts using a UV laser to irradiate a 0.33 µm2 area at maximum power for 15 sec at t = 10 sec. Images were captured every second for 5 min, and the mean fluorescence intensity of FM1-43 was measured on a 13.5 µm2 area around the damage with the Zeiss LSM 800 imaging software.

Zebrafish larvae (6 dpf) were first anaesthetised in Evan’s solution (134 mM NaCl, 2.9 mM KCl, 2.1 mM CaCl₂, 1.2 MgCl₂, 10 mM HEPES, 10 mM glucose) containing 0.02% tricaine (Sigma Aldrich). The larvae were then pinned to a Sylgard-coated dish both at the head and extreme tail end using electrolytically sharpened tungsten needles. The skin was then carefully peeled away to expose the muscles and to permit access to FM1-43 (Molecular Probes). The fish were treated with Evan’s solution containing 10 μM of FM1-43 to allow preloading penetration of the dye molecules. After 10 min, the fish were transferred to a high potassium HBSS (97 mM NaCl, 45 mM KCl, 1 mM MgSO₄, 5 mM HEPES, 5 mM CaCl₂) containing 10 μM of FM1-43 for 5 min. The fish were then incubated with an Evan’s solution with 10 μM of FM1-43 finished for an additional 3 min, after which loading was complete. The larvae were then washed with a low calcium Evan’s solution (0.5 mM CaCl₂) three times for 5 min to minimize spontaneous release of loaded SVs. The fish were imaged for FM1-43 staining using a ×40 Examiner A1 microscope (Zeiss). Blind measurements of FM1-43 staining at NMJs in wild-type control and C9-miR fish were performed per three somites per fish for each genotype using Fiji ImageJ (NIH).