Isolation and Culture of Zebrafish Muscle Fibers

Isolation and culture of zebrafish adult muscle fibres was previously described (Ganassi et al., 2018 (link)). Zebrafish aged 8–9 months (adult) or 1 month (juvenile) were culled in high-dose tricaine (Sigma Aldrich), immersed for 5 min in 1% Virkon (3S Healthcare) diluted in dH₂O, washed in PBS for 5 min followed by 70% ethanol rinse, eviscerated and skinned. Fifteen-month-old adult were used for the myog^fh265 allele. For myofibre dissociation, trunk muscle was incubated in 0.2% Collagenase (C0130, Sigma Aldrich), 1% Penicillin/Streptomycin DMEM supplemented with 50 µg/ml gentamycin (Thermo Fisher) at 28.5°C for at least 2 hours. Single muscle myofibres were released by trituration using heat-polished glass pipettes and washed three times with DMEM Glutamax High Glucose (Gibco). Myofibres were then imaged for total length measure or plated on Matrigel (Invitrogen) coated 96- or 24-well plates and cultured in growth medium (20% Fetal Bovine Serum in 1% Penicillin/Streptomycin/DMEM Glutamax High Glucose supplemented with 10 µg/ml gentamycin). At indicated time points, MPCs were washed twice with PBS to remove plated myofibres, EdU pulsed (10 μM, Invitrogen Life Technologies) for 8 hr in fresh media and then fixed with 4% PFA for 15 min. For immunostaining myofibres were fixed in 4% PFA in PBS immediately after dissociation to reduce processing time and avoid MuSC activation.