Discovery v12 microscope

Terminal deoxynucleotidyl transferase (TdT) nick-end labelling (TUNEL) assay was performed for map11-KO line according to manufacturer’s instructions (Roche; 12156792910). Briefly, chorions of 24 h-post-fertilization (hpf) zebrafish embryos were removed using gentle forceps and fixed with 4% PFA over-night at 4°C. Embryos were washed three times for 10 min each in PBST (0.5% Triton™ X-100 in PBS) for permeabilization and blocked for 1 h at room temperature in blocking solution [0.5% Triton™ X-100, 10% foetal calf serum (FCS), 1% dimethyl sulphoxide (DMSO) in PBS]. Embryos were then labelled using TUNEL reaction mixture (labelling solution with terminal transferase enzyme). Embryos served as positive controls were incubated with 5 U/ml of recombinant DNase I in 50 mM Tris-HCl, pH 7.5, 1 mg/ml bovine serum albumin) for 10 min at room temperature prior to labelling. Embryos served as negative control were incubated without terminal transferase enzyme. After incubation, embryos were washed in PBST and visualized with fluorescent Discovery.V12 microscope (Zeiss). Image analysis was done using ImageJ. Fluorescent intensity was measured by integrated density of whole embryos to calculate relative apoptosis.

Images of mounted wings were captured by Zeiss Axio Imager.M2 with Zeiss Axiocam 503 color camera. Whole adults were imaged using Zeiss AxioCam MRc5 connected to the Zeiss Discovery V12 microscope. Zeiss AxioVision Extended Focus module was used to obtain images with increased focus depth. Some pictures were enhanced only for brightness and contrast with Adobe Photoshop Creative Cloud.