Pa1 094

Histological examination, whole-mount and section immunostaining and ISH were carried out according to standard procedures. Briefly, inner ears were fixed in 4% paraformaldehyde (PFA) for 1 hr at 4°C, dehydrated, and embedded in wax. Paraffin sections were generated at 6 μm. For ISH, tissues were fixed overnight. We used five embryos for each genotype at each stage for each probe and the result was consistent in each embryo.
Primary antibodies: anti-Sox2 (PA1-094, Thermo Fisher), -Myo7A (25–6790, Proteus and 138-1-s, DSHB), -Six1 (HPA001893, Sigma), -Atoh1 (Math1-s, DSHB), -p27^kip1 (554069, BD Pharmingen), -Calretinin (MA5-14540, Thermo Fisher), -p75^NTR (#07–476, EMD Millipore), -N-cadherin (610921, BD Bioscience), -E-cadherin (U3254, Sigma), -S100A (ab11428, Abcam), -GLAST (ab416, Abcam), -Pou4f3 (sc-81980, Santa Cruz), -Prox1 (AB5475, Millipore), -Acetylated tubulin (T7451, Sigma), -Cy3-, Cy2-, Cy5- and FITC-conjugated secondary antibodies were used. Alexa Fluor 488 or 350-conjugated phalloidin (A12379 and A22281, Life technologies) were used for actin staining. Hoechst 3342 was used for nuclear staining.

Histology and immunostaining were performed following standard procedures. Briefly, tongues were collected in ice-cold PBS and fixed in 4% PFA for 30 min at room temperature. Samples were cryoprotected in 30% sucrose at 4°C overnight and then submerged in OCT compound and flash frozen on dry ice. Frontal sections were generated at 6 μm. Some sections were pretreated with citrate buffer (10 mM, pH6.0) at 65°C for 3 h for antigen retrieval before IHC.
Primary antibodies used were before IHC rabbit anti-Sox2 (PA1-094, ThermoFisher), rabbit anti-Six1 (50-204-6211, Cell Signaling), mouse anti-TUBB3 (66375-1-Ig, Proteintech), rabbit anti-β-Gal (08559761, MP Biomedicals), rabbit anti-Pou2f3 (PA5-40556, ThermoFisher), mouse anti-IP₃R3 (610312, BD Biosciences), rabbit anti-Ncam (ab220360, Abcam), and mouse monoclonal Keratin 8 (Krt8) (NBP2-44940, Novusbio). Cy3-or Cy2-conjugated secondary antibodies were used. Nuclear staining was performed using Hoechst 33342.