Zebrafish Pronephros Regeneration Imaging

Laser-induced ablations were performed as previously described [7 (link)]. Briefly, 2-day-old zebrafish larvae were embedded in 1% low melting agarose in glass-bottom dishes. Cell ablations were performed with a 2-photon laser (Chameleon) attached to an LSM 880 Observer confocal microscope (Carl Zeiss, Jena, Germany). In total, 80 µm of the pronephros was ablated. For WISH, larvae were fixed in methanol 2 h post ablation. Confocal images were recorded with a C-Apochromat 40×/1.2 objective (Carl Zeiss, Jena, Germany). Time-lapse video microscopy was carried out at the LSM 880 microscope. Z-stacks of the injury site were recorded every 10 min. The 3D reconstruction, track speed and cell displacement were calculated in Imaris (Bitplane, Zürich, Switzerland). The tubular repair was monitored with a Leica MZ16 epifluorescent stereo microscope (Leica, Solms, Germany).