Clostridium hystoliticum type 2

Twenty-one-week-old WT and dHT vehicle- and drug-treated male mice were killed by pentobarbital overdose according to the procedures approved by the Kantonal Veterinary Authority. FDB muscles were isolated and digested with 0.2% of collagenase type I (Clostridium hystoliticum Type I, Sigma-Aldrich) and 0.2% of collagenase type II (Clostridium hystoliticum Type II, Worthington) in Tyrode’s buffer (137 mM NaCl, 5.4 mM KCl, 0.5 mM MgCl₂, 1.8 mM CaCl₂, 0.1% glucose, 11.8 mM HEPES, pH 7.4 NaOH) for 45 min at 37°C as described (Calderón et al., 2009 (link)). Muscles were washed with Tyrode’s buffer to block the collagenase activity and gently separated from tendons using large to narrowest set of fire-polished Pasteur pipettes. Fibers obtained by this procedure remained excitable and contracted briskly when assayed experimentally. Finally, fibers were placed on laminin coated (5 µl of 1 mg/ml mouse laminin from Thermo Fisher) 35 mm glass bottom dishes (MatTek corporation) for measurements of the resting [Ca²⁺] or on ibiTreat 15µ-Slide four well (Ibidi) for electrically evoked Ca²⁺ measurements as previously described (Elbaz et al., 2019 (link)).