Fluorescent RNA in situ hybridization

Mouse inner ear tissues were collected, total RNA was purified using the RNeasy Plus Mini kit (Cat# 74134, Qiagen Inc., Valencia, CA, USA) combined with extra on-column DNase treatment with an RNase-free DNase set (Qiagen), and first-strand cDNA was generated using SuperScript^® VILO^™ MasterMix (Invitrogen, Grand Island, NY) according to the manufacturers’ instructions. The resultant product was used as PCR template to amplify the cDNA sequences for generating RNA probes for in situ hybridization. Primers 5’-ACACTCGAGCTGTGAGAAAAAAGATCC-3’ and 5’- ACATCTAGAGAAACGCCAGTCAGTG-3’ were used to amplify a 109bp sequence for detecting mouse Pcdhga10 long isoform, and primers 5’-ACACTCGAGCTGTGAGAAAAAAGATCC-3’ and 5’-ACATCTAGATTTGGGCTCAAGCACAACG-3’ were used to amplify a 143bp sequence for detecting a mouse Pcdhga10 short isoform that is at the equivalent base position of human PCDHGA10.

Amplification products were purified using QIAquick PCR purification kit (Qiagen) and cloned into pGEM-T easy vector (Promega, Madison, WI). X-Gal/IPTG-selected clones were digested with restriction enzymes to confirm the size of inserts, then sequenced for further confirmation as well as determination of the orientation. The confirmed clones were linearized with XhoI or XbaI, and used to generate digoxigenin‐labeled single‐strand antisense and sense RNA probes for hybridization using the DIG RNA Labeling Kit (SP6/T7) (Cat# 11175025910, Roche Molecular Biochemicals, Alameda, CA) according to the manufacturer’s instructions.

Animals were deeply anesthetized with ketamine-xylazine (ketamine: 200 mg/kg; xylazine: 5 mg/kg body weight) and then decapitated. All following steps were carried out under RNase-free conditions with diethyl pyrocarbonate (DEPC)-treated solutions or buffers made with DEPC-treated water. Inner ears were dissected in PBS and fixed in 4% paraformaldehyde (PFA) in PBS for 4 hours at room temperature, decalcified in 0.25 M EDTA (pH7.4) overnight, dehydrated in 30% sucrose prepared in PBS, embedded in optimal cutting temperature (O.C.T) compound at below -20°C, sectioned at 9 μm using a MICROM HM-505 N cryostat (Microm, Germany), thaw-mounted on Superfrost Plus glass slides (Thermo Fisher Scientific) and air-dried. The sections were stored at -80°C until processed for fluorescent in situ hybridization (FISH).

Frozen tissue sections were warmed to room temperature and desiccated for 20 min at 50°C, post-fixed in 4% PFA, followed by two washes in PBS. Then sections were treated with 5 μg/ml proteinase K (Thermo Fisher Scientific) in 50 mM Tris buffer, pH 8.0, containing 5 mM EDTA, for 5 min at 37°C. After two washes in 2X saline sodium citrate (SSC), sections were acetylated with 0.1 M triethanolamine, pH 8.0, containing 0.25% (v/v %) acetic anhydride for 20 min, and rinsed in 2X SSC. Sections were treated with pre-warmed pre-hybridization solution composed of 50% formamide, 4X SSC, 10% Dextran sulfate, 1X Dernhardt’s and 50 μg/ml yeast tRNA (Sigma-Aldrich, St. Louis, MO, USA) in an RNase-free humid chamber containing a thin layer of 3M Whatman paper soaked with 4X SSC/50% formamide for 2 h at 55°C. The pre-hybridization solution was then replaced with pre-warmed hybridization buffer containing 0.3 mg/ml sheared, denatured herring sperm DNA and 0.5 μg/ml digoxigenin-labeled anti-sense/sense RNA probe, and the tissue sections with cover slips were incubated overnight at 55°C in the humid chamber.

Following hybridization, sections were rinsed in 2X SSC to wash-off the cover slips and treated with 20 μg/ml RNase A (Sigma-Aldrich) in 10 mM Tris, pH 8.0, containing 500 mM NaCl and 1 mM EDTA, for 30 min at 37°C, followed by additional stringent washes in 2X SSC/50% formamide (5 min), 1X SSC (5 min), 0.5X SSC (5 min), at 50°C and three washes in PBS at room temperature. After that, sections were stained using the Alexa Fluor^™ 488 Tyramide SuperBoost^™ Kit (Cat# {"type":"entrez-nucleotide","attrs":{"text":"B40922","term_id":"2545174","term_text":"B40922"}}B40922, Thermo Fisher Scientific) combined with rabbit-derived monoclonal digoxigenin antibody (Cat# 700772, Thermo Fisher Scientific) according to the manufactures instructions. Briefly, sections were blocked with 10% normal goat serum for 60 min at room temperature, and incubated with digoxigenin antibody (1:500 in blocking buffer) overnight at 4°C, followed by three washes in PBS. Sections were then incubated with poly-HRP-conjugated goat anti-rabbit secondary antibody (1: 600 in blocking buffer), together with DAPI (Sigma-Aldrich, St. Louis, MO, USA) at a dilution of 1:10,000, in the dark for 60 min at room temperature. After three washes in PBS, for the signal enhancement, the tyramide working solution was applied to the sections for 6 min at room temperature, and the stop solution was used to terminate the HRP reaction. After three more washes in PBS, sections were mounted in Fluoromount-G and fluorescence images were acquired using a Zeiss Axio Observer Z1 inverted microscope equipped with an AxioCam MRm camera and with GFP, DsRed and DAPI filter sets.