Rabbit anti 5 ht

Larval fish at 4 dpf, 5 dpf and 6 dpf were fixed overnight at 4°C in 4% PFA, rinsed three times in 1X PBS, incubated in 100% Methanol at −20°C for 1 hour and then rehydrated step-wise into 1X PBS at room temperature. Larvae were then incubated in 100% acetone at −20°C for 11 minutes, rinsed three times in 1X PBS, then digested at room temperature in 10 μg/mL Proteinase K for 45 min. (4 dpf), 55 min. (5 dpf) or for 65 min. (6 dpf). Larvae were then rinsed three times in 1X PBS, fixed for 10 minutes in 4% PFA at room temperature, rinsed 3 times in 1X PBS and then incubated in 5% Donkey serum block diluted in 1X PBS-tween-20, supplemented with 1% DMSO (PBTD), for 3 hours. Embryos were then incubated in either rabbit anti-GFP 1:500 (Life Technologies, A-11122), goat anti-GFP 1:500 (Abcam, ab6673), mouse anti-HuC/D (Hu) 1:200 (Invitrogen), mouse anti-Acetylated tubulin 1:1000 (Sigma, T6793) or rabbit anti-5HT 1:1000 (Immunostar) overnight at 4°C. Embryos were then washed out of primary antibody in 1X PBS-tween-20, then incubated at room temperature in 1:700 secondary antibodies Invitrogen Alexa Fluor Donkey anti-Rabbit 488, anti-Rabbit 647, anti-Goat 488 or Donkey anti-Mouse 594 for 3 hours at room temperature. Embryos were rinsed in 1X PBST and imaged in 75% glycerol/1X PBS on a Zeiss 710 2-photon confocal microscope (Beckman Imaging Center, Caltech).

IHC and ISH were performed as described (Zhao et al., 2006 (link)). For the IHC study, sections were incubated with primary antibodies overnight at 4°C followed by the use of FITC or Cy3-conjugated secondary antibodies (Jackson ImmunoResearch). The following primary antibodies were used: rabbit anti-5-HT (1:5,000, Immunostar), rabbit anti-5-HT-1A (1:200, Santa Cruz) and chicken anti-GFP (polyclonal, 1:500, Aves Labs). For the ISH study, a digoxigenin-labeled cRNA probe was used as described earlier (Zhao et al., 2006 (link)). Images were taken using a Nikon Eclipse Ti-U microscope.

Both wholemounts and sections were incubated for 24–48 hours at 4°C with primary antibodies, followed by 24–48 hours at 4°C with the appropriate secondary a ntibodies (Molecular Probes, Invitrogen). The blocking solutions used were PBS/ 0.5% Triton X-100/ 10% Normal Donkey Serum (NDS) for wholemounts, and PBS / 0.1% Triton X-100/ 5% NDS for sections. For BrdU staining, samples were incubated for 40 min at 37°C w ith 2N HCl followed by 10 min at 25°C with 0.1 M boric acid (pH8.5), prior to incubation with antibodies. For mounting, the wholemounts and sections were embedded with Aqua Poly/Mount mounting medium (Polysciences). Primary antibodies: Mouse anti-AcTub (Sigma, 1:1000), Rabbit anti-5HT (Immunostar, 1:500), Rabbit anti-SERT (Immunostar, 1:500), Rabbit anti-synaptophysin (Abcam, 1:500), Goat anti-vGluT3 (Abcam, 1:200), Rabbit anti-ACIII (Santa Cruz, 1:500), Mouse anti-β-catenin (BD Biosciences, 1:500), Chicken anti-GFP (Aves Labs, 1:500), Rabbit anti-DCX (Cell Signaling, 1:200), Mouse anti-GFAP (Millipore, 1:1000), Mouse anti-Mash1 (BD Biosciences, 1:200) and rat anti-BrdU (Abcam, 1:500).