Anti huc hud

To determine the proportion of terminally differentiated enteric neurons, 5 dpf F2 zebrafish larvae (tfap2b^+/-, phox2bb:GFP) treated with PTU were immunostained with antiHuC/HuD, a mature neuronal marker. Larvae were incubated on ice for 30 min before being fixed in 4% PFA and washed in 1x phosphate buffer solution/0.25% Triton X-100 for 1 h, at room temperature. Whole mount antibody staining was performed according to previous reports (Uyttebroek et al., 2010 (link)). Anti HuC/HuD (1:100, A-21271, Invitrogen, Waltham, Massachusetts, United States) was used as primary antibody and Cy3 Mouse IgG (1:500, Thermo Fisher Scientific, Waltham, Massachusetts, United States) as secondary antibody. Larvae were imaged under the confocal microscope (Leica SP5 AOBS, Leica Camera, Wetzlar, Germany). The number of phox2bb:GFP⁺ and HuC/HuD⁺ cells were counted using Fiji ImageJ software. The proportion of differentiated enteric neurons was determined by calculating the ratio of HuC/HuD⁺ cells to phox2bb:GFP⁺ cells. All larvae were genotyped at the end of the experiment.

Seven dpf WT and mutant larvae were incubated on ice for 30 min before being fixed in 4% Formaldehyde (Pierce^TM, Thermo Fisher Scientific, 28906)-1× phosphate buffer solution/0.25% Triton X-100 (1× PBSTx) for 1 h at room temperature. Whole-mount antibody staining and gut dissections were carried out according to previously published methods [60 (link)]. Anti-HuC/HuD (1:1000; Invitrogen; A21271) and anti-5-HT (1:1000; serotonin; Immunostar, New Richmond, WI; 20,080) were used as primary antibodies with secondary antibodies conjugated to Alexa Fluor 568 (1:1000; Abcam, ab175472) and Alexa Fluor 633 (1:1000; Thermo Fisher Scientific, A-31575). To sample the anterior and posterior intestinal tract, a 200 × 200-μm field was captured 200 μm from the intestinal bulb (anterior) and 200 μm from the anus (posterior) as in [61 (link)]. Images were captured using a 20× dry objective and analyzed using the cell counter application in Fiji.