Acetylated tubulin

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.

Animals were fixed with AB fix [4% Paraformaldehyde and 1×PBST (1% TritonX)] for 3 h at 23°C and then washed in 1×PBST (5% TritonX), ddH20Tx (5% TritonX), and acetone for 5 min each at 23°C and then an additional acetone wash at −20°C. 1×PBST (5% TritonX) with 5% goat serum was used to block for at least an hour. Animals were incubated in primary antibody overnight at 4°C. The primary antibodies used in this study include the following: Sox10, 1∶5,000 [49] (link); Acetylated Tubulin, 1∶10,000 (Sigma); HuC, 1∶100 (Invitrogen); MBP, 1∶250 [27] (link). Animals were washed extensively with 1×PBSTx before the secondary antibody was added. These antibodies include Alexa antibodies (1∶600): goat anti-rabbit 568, goat anti-mouse 568, goat anti-rabbit 647, and goat anti-mouse 647. After extensive washes, animals were stored in 50% glycerol/50% 1×PBS until imaged and mounted under a bridged coverslip.

NIH3T3 (ATCC CRL-1658) or HEK-293 cells (ATCC CRL-1573) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS), 1000 U/ml penicillin, 1000 μg/ml streptomycin, and 20 mM L-Glutamine (all Gibco) at 37°C and 5% CO₂. NIH3T3 cells were grown on coverslips in 6-well plates and transfected using EndoFectin^TM Max Transfection Reagent, following the recommendations of the manufacturer (GeneCopoeia). Twenty-four hours after transfection, cells were washed in phosphate-buffered saline (PBS) and fixed in methanol for 10 min at −20°C. Specimens were then permeabilized in 0.3% TritonX-100 in PBS for 10 min at room temperature and blocked for 1 h using blocking solution (PBS containing 1% BSA and 0.3% TritonX-100). Samples were incubated with primary antibodies toward CCDC42 (ARP52735_P050, antibodies-online ABIN2785068), Pericentrin (PRB432C, Covance), acetylated tubulin (6-11B-1; Sigma-Aldrich), gamma-tubulin (GTU-88, Sigma-Aldrich), ODF1 (ABIN4341345, antibodies-online), and GFP (raised in rabbit, self-made) at 37°C for 1 h. Secondary antibodies used are goat anti-mouse-IgG DyLight 488 (#35503, Thermo Fisher Scientific), and goat anti-rabbit MFP590 (#MFP-A1037, Mobitec). DNA was counterstained with DAPI. Images were taken by confocal microscopy (LSM 780, Zeiss) and processed using Adobe Photoshop 7.0.