Pharmacological immobilization in vivo

Dechorionated wild-type embryos were transferred into Embryo Medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl₂, and 0.33 mM MgSO₄, pH 6.8–6.9) supplemented with 0.03% Tricaine (Tricaine methanesulfonate, TMS, MS-222) at 17 hpf, incubated for 7 h at 28.5°C. After 1 h of treatment embryos were examined for locomotor activity, i.e., occurrence of spontaneous movements. Control embryos, treated in exactly the same way as experimental group in Embryo Medium but without Tricaine, were raised in parallel. At 24 hpf, control and treated embryos were fixed and stained immediately or put into recovery (Embryo Medium without Tricaine). The control and recovered embryos were either fixed and stained at 42 hpf or mounted for X-ray diffraction at 5 dpf, or mechanically assessed at 7 dpf or used to assess their swimming behavior (at 3, 5, 7, and 30 dpf). Experiments to measure sarcomere length at different degrees of relative stretch included a group of larvae that were immobilized from 17 hpf to 5 dpf. Embryos were incubated initially at 28.5°C in 0.03% Tricaine from 17 hpf up to 29 hpf, and subsequently the larvae were transferred into 50 μM BTS (N-Benzyl-P-Tuloenesulfonamide, Sigma) and further incubated at 28.5°C up to 5 dpf.