Tricaine

For photos of live embryos at 30 hpf, the embryos were embedded in 2% methylcellulose (Sigma-Aldrich) in embryo medium with 0.04% tricaine (Sigma-Aldrich). For time-lapse vital microscopy, embryos were embedded in 1% low-melting agar in embryo medium with 0.04% tricaine (Sigma-Aldrich). When imaging the periderm, recording started at 28 hpf and continued for 18 h, with images acquired at 8-min intervals. A 710 laser-scanning confocal (Zeiss) and spinning disk fluorescence (Nikon) microscopes were used. When imaging the basal layer, 2-h recordings were performed between 24 and 30 hpf with images collected at 2-min intervals. A 710 confocal fluorescence microscope (Zeiss) was used. Imaging was performed with a long working distance 20× lens. All the time-lapse studies were performed at 28.5°C. Image analysis was performed using Zeiss Black or Fiji ImageJ software.

Wild-type AB (WT) and heterozygous Chihuahua (col1a1a^dc124/+, Chi/+) zebrafish arise from natural spawning or from in vitro fertilization. The mutant Chi/+, provided by Professor Shannon Fisher (Dept of Pharmacology & Experimental Therapeutics, Boston University School of Medicine, USA), carries a G2207A mutation in col1a1a, causing a p.G736D (G574D) substitution in the α1 chain of type I collagen. Embryos were kept in Petri dishes in fish water (NaHCO₃ 1.2 mM, instant ocean 0.1 g/L, CaSO₄ 1.4 mM, methylene blue 0.00002% w/v) at 28 °C until 6 day post-fertilization (dpf), then housed in ZebTEC semi-closed recirculation housing systems (Tecniplast) and kept at a constant temperature (27–28 °C), pH (7.5) and conductivity (500 μS) on a 14/10 light/dark cycle. For the experiments stated below larvae and adult fish were anesthetized using a solution of tricaine (3-amino benzoic acidethylester, Sigma Aldrich) 0.0016% w/v, in fish water and sacrificed by tricaine overdose (0.03% w/v). All experiments were performed in accordance with the approved guidelines, in agreement with EU Directive 2010/63/EU for animals. The experimental protocol was approved by the Italian Ministry of Health (Approval Animal Protocol No. 1/2013).

Cryoinjury was performed as previously described (González-Rosa and Mercader, 2012 (link)). Adult fish were anesthetized with 0.032% tricaine (Sigma, St Louis, MO, USA) and their pericardial cavity opened with microdissection scissors to expose the heart. A copper filament cooled in liquid nitrogen was placed on the ventricular surface of the heart until thawing. After surgery, animals were revived by gently directing water to their gills using a plastic Pasteur pipette.
For analysis of regeneration, animals were euthanized at different times post-injury by immersion in 0.16% tricaine (Sigma, St Louis, MO, USA), and hearts were dissected in media containing 2 U/ml heparin and 0.1 M KCl. For quantification of injured area on paraffin sections as shown in Figures 1F and 1G, color deconvolution tool and color threshold tool (ImageJ Software) were used to segment and measure the injured and uninjured myocardium in μm².