Tailfin transection, fixation and staining

3 dpf larvae were anaesthetised by the addition of 0.02% buffered 3-aminobenzoic acid ethyl ester (Tricaine/MS-222) into the embryo medium and were left until paralysed. Using a scalpel, the entire tail fin and a small portion of the trunk distal to the end of the vasculature was removed. The embryos were then placed in fresh medium and allowed to recover. At 2 hours post injury (hpi), 6 hpi and 22 hpi larvae were culled using excess Tricaine. Culled larvae were then placed in an Eppendorf containing 4% PFA, 0.4% Triton-X diluted in PBS and fixed overnight at 4°C or at room temperature for 2 hours.

Wholemount immunostaining of zebrafish larvae was performed as described previously³⁸. Wash buffer comprising PBS containing 0.1% Triton-X (PBST Sigma-Aldrich) and 5% horse serum (Sigma Aldrich) was used for blocking. Both primary and secondary antibodies were diluted in PBST containing 2%–5% horse serum and were left to incubate over night at 4°C. DAPI was added to secondary antibodies to visualize the entire tissue. Primary antibody: α-mCherry (1:500, Abcam {"type":"entrez-nucleotide","attrs":{"text":"Ab125096","term_id":"76879953","term_text":"AB125096"}}Ab125096). Secondary antibody: α-mouse AF 568 (1:250 Invitrogen A21124). Stained samples were mounted laterally in Vectashield on glass slides and imaged on a Zeiss LSM880 confocal microscope using a 25x objective (NA 0.8).