B 8503

Frzb in situ hybridization probe was kindly provided by Prof. De Robertis (Leyns et al., 1997 (link)). The labeled probe was ethanol-precipitated, resuspended in 100 mM DTT, diluted in hybridization solution (60% deionized formamide, 20 mM Tris-HCl, 5 mM EDTA, pH 8, 0.3 M NaCl, 0.5 mg/ml yeast RNA, 5% dextran sulfate). In situ hybridization was performed according to standard procedures (Mitsiadis et al., 1995 (link)). Briefly, slides were incubated with the probe at 60°C. After intense washing, the slides were incubated in blocking solution (20% Normal Goat Serum) and anti-digoxigenin (DIG)-AP (alkaline phosphatase conjugate) Fab-fragment (Boehringer Mannheim, 1093 274) diluted 1:1,000 in blocking solution. The color reaction was developed using Nitro Blue Tetrazolium (NBT, Sigma N-6876) and 5-Bromo-4-Chloro-3-Indolyl Phosphate (BCIP, Sigma B-8503) in staining solution 2% NaCl, 5% MgCl2, 10% Tris-HCl pH 9.5, 1% Tween-20. In situ hybridization immediately followed by BrdU immunohistochemistry was performed in cryosectioned slides of E13–E15 mouse embryos to show the correlation between Frzb expression and cell proliferation (Mitsiadis et al., 2008 (link)). No hybridization signal was detected with the sense probe at these developmental stages.

Zebrafish in situ hybridization experiments were performed according to the standard procedure for in situ hybridization reported previously [32 (link)]. The T7 promoter sequence was added to the 5’ end of the reverse primer used to synthesize the antisense RNA probe template for PCR amplification (Q5^® Hot Start High-Fidelity 2× Master Mix, M0494, NEB, Ipswich, MA, USA) with a cDNA template from 72 hpf wild-type zebrafish embryos. Digoxigenin-labelled antisense RNA probes were synthesized using T7 RNA polymerase (EP0111, Thermo, Waltham, MA, USA) and DIG RNA Labeling Mix (11277073910, Roche, Basel, Switzerland). After 24 hpf, the embryos were permeabilized with proteinase K: 24 hpf (2 min), 48 hpf (30 min), 72 hpf (45 min). DIG-labelled probes were detected using an alkaline-phosphatase-conjugated anti-DIG Fab fragments (11093274910, Roche, Switzerland), followed by staining with nitro blue tetrazolium (NBT; N6876, Sigma, Burlington, MA, USA) and 5-bromo-4-chloro-3-indolyl phosphate (BICP; B8503, Sigma, USA). All primer sequences used for the synthesis of antisense RNA probes are listed in Supplementary Table S1.