Buffered 3 aminobenzoic acid ethyl ester tricaine

Zebrafish lines in this study (S1 Table) were handled in compliance with local animal welfare regulations as overseen by the Animal Welfare Body of Leiden University (License number: 10612) and maintained according to standard protocols (zfin.org). All protocols adhered to the international guidelines specified by the EU Animal Protection Directive 2010/63/EU. The generation of zebrafish optn^ibl51 and p62^ibl52 mutant lines was approved by the Animal Experimention Committee of Leiden University (UDEC) under protocol 14198. All experiments with these zebrafish lines were done on embryos or larvae up to 5 days post fertilization, which have not yet reached the free-feeding stage. Embryos were grown at 28.5°C and kept under anesthesia with egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (Tricaine, Sigma) during bacterial injections, imaging and fixation.

CRISPR-Cas9 technology was used to generate a galanin mutant (gal−/−) as described [41 (link)]. The mutant has a 10-base-pair deletion in the 3rd exon of the gal gene (Ensembl (GRCz11): ENSDARG00000091377), which results in a loss-of-function allele due to a frameshift and premature stop codon. A detailed description of this mutant is in preparation for a separate publication (unpublished data).
The wild-type gal+/+ and gal−/− mutant larvae were maintained as described previously [42 (link)]. Embryos were raised in E3 medium (5 mM NaCl. 0.17 mM KCl. 0.33 mM CaCl₂. 0.33 mM MgSO₄) at 28.5 °C. During imaging, fish were kept under anesthesia in egg water containing 0.02% buffered 3-aminobenzoic acid ethyl ester (Tricaine, Sigma-Aldrich, St. Louis, MO, USA).
All fish lines are housed both in the fish facility of the Laboratory of Genomics and Transcriptomics, University of Warmia and Mazury in Olsztyn, Poland, which was built according to the local animal welfare standards and in the fish facility of Leiden University compliant with the directives of the local animal welfare committee. Studies performed on early-life-stage zebrafish larvae and euthanasia do not require Ethic Committee permissions. According to the European Directive 2010/63/EU and Polish law regulations O.J. of 2015, item 266.