Mesab

For immunostaining, embryos were treated with phenyl thiourea (PTU, 0.2 mM) to prevent pigmentation from 22 hpf on. Larvae were sacrificed with an overdose of MESAB (ethyl 3-aminobenzoate methanesulfonate, Sigma-Aldrich), and fixed with ice-cold 4% PFA in PBS overnight at 4°C. The next day, samples were washed with PBS with 0.1% Tween-20 (PBST) and processed through 10%, 20% and 30% sucrose solutions. Samples were incubated in 1:1 30% sucrose, 50% NEG-50 (Thermo Fisher Scientific) and then embedded in NEG-50 and stored at −80°C. For adult eyes, zebrafish were sacrificed with an overdose of MESAB and decapitated. Zebrafish eyes were dissected and fixed in 4% PFA overnight at 4°C. After fixation, samples were washed 2×5 min in PBS and kept for 1 h in 5% sucrose in 1×PBS, and then incubated overnight at 4°C in 30% sucrose in 1×PBS, followed by a second overnight incubation at 4°C in 1:1 solution of 30% sucrose in 1×PBS/NEG-50™ (Thermo Fisher Scientific). Finally, the samples were incubated for 1 h at room temperature (RT) in NEG-50™, mounted and frozen in dry ice. All samples were kept at −80°C until sectioning. Cryosections were generated using a Microm HM 560 (Thermo Fisher Scientific) at 16–20 µm, and left to dry for at least 2 h at room temperature. After sectioning, all samples were kept at −20°C until use.

Embryos were anaesthetised in MESAB (E10521 Sigma), the flank and the median fin fold was chopped and total RNA was extracted using Trizol (Invitrogen). cDNA was synthesised using Cloned AMV-RT-kit (Invitrogen). Quantitative RT-PCR was carried out in AB Step Plus RT PCR system using appropriate primers (see methods in the supplementary material). The splicing efficiency for different doses of laminin α5 MO was estimated as described previously (Webb et al., 2007 (link)) and actin transcript levels were used as control for normalisation.

PA-GFP positive larvae were raised in a dark incubator to prevent background photoconversion. Larvae were anesthetized with 0.2 mg/mL ethyl-3-aminobenzoic acid ethyl ester (MESAB, Sigma-Aldrich E10521, St. Louis, MO) and mounted laterally in 2% low-melting temperature agarose (Thermo Fisher Scientific 16520). Using a Zeiss LSM800 confocal microscope with 20x objective (Zeiss W Plan-Apochromat 20x/1.0 DIC CG=0.17 M27 75mm), the spinal cord between the mid-point of the swim bladder and the caudal-most tip of the tail was repeatedly scanned with a 405 nm laser until fully converted. For retrograde labelling of vestibulospinal neurons used for photoablations, the spinal cords of Tg(α-tubulin:C3PA-GFP) larvae were converted at 6 dpf. To allow the converted fluorophore to diffuse into neuron bodies, all fish were removed from agarose after photoconversion and raised in E3 in a dark incubator.

Adult Zebrafish retinas were collected, anesthetized using MESAB (0.2% ethyl‐m‐aminobenzoate methanesulfonate) (Sigma‐Aldrich), and killed by cutting their heads. Retinas were dissected using forceps and fixed in 4% paraformaldehyde (PFA) in phosphate‐buffered saline (PBS) for 2 hr at room temperature. Retinas were then cut, placed on a drop of Vectashield antifade mounting medium (Vector Labs) on top of a glass slide, and flattened using a coverslip.