C apochromat 40 1.2 objective

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).

Cell ablation was performed using 2-photon laser (Chameleon) mounted on LSM 510 Zeiss microscope (Carl Zeiss, Jena, Germany). Approximately 80 µm of the pronephros were ablated unless stated otherwise. Embryos were analyzed under a Leica MZ16 stereo-microscope (Leica, Solms, Germany). Differential interferences contrast (DIC) and non-confocal fluorescent images were taken with a SPOT Insight Fire Wire System (Diagnostic Instruments, Sterling Heights, USA). For time-lapse movies the embryos were embedded in 1.5% low melting agarose. Confocal images were recorded on Zeiss LSM 510 with a C-Apochromat 40×/1.2 objective (Carl Zeiss, Jena, Germany). Confocal Z-stakes were acquired every 3 min, and time-lapse movies from the maximum Z-projections were assembled using LSM, ImageJ or Imaris software. Cell tracking was performed using Imaris software. In all movies, the cranial side of the embryo is on the left side.

A total of 80 µm of thee pronephric tubule of 2-day-old zebrafish larvae were ablated as previously described [7 (link),8 (link)]. Cell ablations were performed with a 2-photon laser (Chameleon) connected to an LSM 880 Observer confocal microscope (Carl Zeiss, Jena, Germany). The repair status was monitored and quantified 24 hpi on a Leica MZ16 epifluorescent stereo microscope (Leica, Solms, Germany). For time-lapse movies, confocal Z-stacks were recorded every 10 min with a C-Apochromat 40×/1.2 objective (Carl Zeiss, Jena, Germany) on the LSM 880 microscope. Z-stacks of the injury site were recorded every 10 min. Three-dimensional reconstruction and time-lapse movie export was carried out in Imaris (Bitplane, Zürich, Switzerland).