Anti 5 ht

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.

All animals were fixed in 4% PFA (in 1X PBS, 0.1% Tween20) for 30 min. Larvae 48 h and older were first relaxed with 1:1 MgCl₂-seawater for 3–5 min before fixing them in 4% PFA (in 1X PBS, 0.1% Tween20) for 30 min. After fixation, the samples were washed two times in PTW, followed by two washes in THT (0.1 M Tris pH 8.5, 0.1% Tween20). Blocking was in 5% sheep serum in THT for 1 h before incubating in primary antibodies (Monoclonal anti-acetylated α-tubulin, 1:300 Sigma product number T6793; Anti-5-HT, 1:500, Immunostar product number 20080; Anti-FMRFamide, 1:500, Immunostar product number 20091) for 48 h at 4 °C. The samples were then subjected to two 10 min washes in 1 M NaCl in THT followed by five 30 min washes in THT before incubating in secondary antibodies (Alexa Fluor 1:500, Thermo Fisher Scientific) overnight at 4 °C. Next, the samples were washed in THT, two 5 min washes followed by five 30 min washes. Specimens were stored in embedding medium (90% glycerol, 1x PBS, and 2% DABCO) at 4 °C.

Larval fish were fixed overnight at 4 °C in 4% paraformaldehyde/1xPBS, after which they were dehydrated through an ethanol series and stored at −20 °C until use.
In situ hybridizations were performed as previously described25 (link). hcrt, npvf and gpr147 ORFs was cloned in a pCS2+ vector using zebrafish cDNA and antisense DIG labelled probes were transcribed using the linearized pCS2+ plasmid containing the ORF. In situs were visualized with Fast Red (Roche) as substrates.
Immunohistochemical stainings were performed as previously described26 (link), using either anti-GFP (1/1000, Torrey Pines Biolabs), anti-mCherry (1/200, Abcam), anti-5HT (1/1000–1/200, ImmunoStar) and anti-NPVF (1/200, Abcam) as primary antibodies and Alexa 488, Alexa 555, Alexa 594, or Alexa 657-conjugated goat anti-rabbit IgG, goat anti-mouse IgG, or donkey anti-rabbit IgG (1/1000–1/200) as secondary antibodies (Molecular Probes).