In Situ Hybridization of Fgf8 in Cochlea

In situ hybridization was performed as described previously [75] (link), with the following modifications. Briefly, antisense RNA probes were generated from cDNA-containing plasmids and labeled with digoxigenin by in vitro transcription, using the DIG RNA labeling kit (Roche Applied Science, Cat# 11175025910). Whole-mount in situ hybridization was performed on mutant and corresponding control cochleae at various ages (P0, P3, P8, and P10). The 2% paraformaldehyde–fixed cochleae were digested briefly with 10 mg/mL of proteinase K for 5–10 min. Cochleae were hybridized with the Fgf8 probe overnight at 60°C in the hybridization solution. After washing, cochleae were incubated overnight at 4°C with an anti-digoxigenin antibody conjugated with alkaline phosphatase (Roche Applied Science, Cat# 11093274910). After several washes, cochleae were incubated in the dark with NBT/BCIP (BM purple substrate, Roche Applied Science, Cat# 11442074001). After periodic monitoring, the reaction was stopped by washing several times with KTBT. The organs of Corti were flat mounted and imaged.