RNA probes and in situ hybridization procedure

The chick Fgf10 probes were the same as used previously ({"type":"entrez-nucleotide","attrs":{"text":"NM_204696.1","term_id":"45382694","term_text":"NM_204696.1"}}NM_204696.1; Sánchez-Guardado et al. 2013). For the chick Fgf3 plasmid, we used BamH1 and T3 enzymes to generate the antisense probes ({"type":"entrez-nucleotide","attrs":{"text":"NM_205327","term_id":"46048689","term_text":"NM_205327"}}NM_205327; Patxon et al. 2010). For the chick Fgf16 plasmid, we used Xba1 and Sp6 enzymes to generate the antisense probes ({"type":"entrez-nucleotide","attrs":{"text":"NM_001044650","term_id":"113206093","term_text":"NM_001044650"}}NM_001044650; Chapman et al. 2006). All riboprobes were labeled with digoxigenin-11-UTP (Roche, Mannheim, Germany) according to the manufacturer’s instructions. In situ hybridization was performed on cryosections following the methods described by Sánchez-Guardado and co-workers (2009, 2013). The sections were post-fixed with 4% paraformaldehyde in PBS for 10 min and then rinsed with PBS for 15 min. The sections were acetylated in a solution containing 234 ml of H₂O-d, 3.2 ml of triethanolamine (Sigma), 420 ml of 36% HCl, and 600 ml of acetic anhydride. After acetylation, the sections were permeabilized in 1% Triton X-100 for 30 min, and then pre-hybridized at room temperature for 2 h in a solution containing 50% formamide, 10% dextran sulfate (Sigma), 5× Denhardt’s solution (Sigma), and 250 mg/ml t-RNA (Roche), in salt solution. Hybridization was performed with 200–300 ng/ml of the probe in the same hybridization solution overnight at 72°C. After hybridization, the sections were rinsed with 0.2% SSC at 72°C for 1–2 h, and then twice with a solution containing 100 mM NaCl and 100 mM Tris-HCl (pH 7.5). After treatment with 10% normal goat serum (NGS) in the same solution for 2 h, the sections were incubated overnight with alkaline phosphatase-conjugated anti-digoxigenin Fab fragments (Roche, 1:3500). The sections were rinsed twice with the same buffer, and then incubated in 100 mM NaCl, 50 mM MgCl₂, and 100 mM Tris–HCl (pH 9.5). The colouring reaction was developed with NBT and BCIP (Roche). The sections were rinsed with PBS and coverslipped with Mowiol (Calbiochem, Bad Soden, Germany). No signal was obtained with the sense probes.