Zen pro 2012

2 dpf morpholino-injected WT AB and mpx:mCherry or 3 dpf WT and mydgf^−/− mpx:mCherry zebrafish larvae were wounded by tail transection or thermal injury, or injected with zebrafish MYDGF protein in the otic vesicle, as described above. Larvae were fixed in 4% PFA at 4°C at 1 and 6 hpw (following tail transection), 3 and 24 hpb (following burn wound) or 2 h after otic vesicle injection. Caudal fins were imaged in PBS at room temperature on a Zeiss Zoomscope (EMS3/SyCoP3; 1× Plan-NeoFluar Z objective; Zeiss) with an Axiocam Mrm charge-coupled device camera using ZenPro 2012 software (Zeiss). For protein injection assays, images of the otic vesicle region were acquired in PBS at room temperature on a spinning-disk confocal (CSU-X; Yokogawa) on a Zeiss Observer Z.1 inverted microscope and an electron-multiplying charge-coupled device Evolve 512 camera (Photometrics), with a Plan-Apochromat 20×/NA 0.8 air objective (5-µm optical sections, 2,355 × 512 resolution) using ZenPro 2012 software (Zeiss). Neutrophil numbers were counted manually in z-projected images using Zen 2.3 Lite software (Zeiss). Neutrophil numbers were counted in the area distal to the tip of the notochord (tail transections), the area distal to the caudal vessel loop (burn wounds), or within the confines of the otic vesicle (protein injections).

For immunostaining of cells in culture, cells grown on glass coverslips (VWR) were washed in PBS, fixed in 4% paraformaldehyde in PBS, and permeabilized in 0.01% digitonin or 0.1% Triton X-100. For ear lesions from P80 Krt17^+/+ or Krt17^−/− Gli2^tg/+ mice, tissues were fresh-frozen in Tissue-Tek O.C.T. (Sakura) and sections were cut at 5 µm. Samples were blocked in 5% normal goat serum (NGS) in PBS for at least 1 h before staining with primary antibodies diluted in blocking buffer for 16 h followed by Alexa Fluor 488– or Alexa Fluor 594–conjugated goat anti–mouse or goat anti–rabbit secondary antibodies (Invitrogen) for 1 h. 1 µg/ml of Hoechst 33342 was used to stain the nuclei, and coverslips were mounted on microscope slides with mounting media containing 1,4-diaza-bicyclo[2.2.2]octane (Electron Microscopy Sciences). Fluorescence images were obtained using the Zen Pro 2012 software (Carl Zeiss) with an Axio Observer.Z1 fluorescence microscope (Carl Zeiss) equipped with an AxioCam MRm camera (Carl Zeiss) at 37°C under a 63× Plan-Apochromat oil immersion lens (Carl Zeiss). The images were processed using Photoshop software (Adobe) to split and pseudo-color individual channels.