SYTOX Green (Invitrogen) diluted 1:1000 in ddH2O for 159, rocking at 4uC. The sections were rinsed with ddH2O, then with PBS for 159, and mounted under a cover glass using hard set Vectashield (Vector Laboratories) for fluorescence without DAPI. Cover glass was sealed with nail polish to prevent evaporation. Sections were imaged on a Zeiss Axiophot 2 confocal microscope (Carl Zeiss), and comparison images were identically post- processed using Adobe Photoshop (Adobe Systems). Western Blotting and Immunoprecipitation Triton X-100-soluble and SDS-soluble proteins from adult donor human, mouse, and rat retinas were isolated following whole retinal homogenization, and quantified by BCA assay. Twenty micrograms of total protein was boiled and loaded per lane, electrophoresed, and transferred to a nylon membrane. The membrane was blocked with 2% BSA in PBT, incubated in primary antibody (CLRN1-B2) at 1:3000 (0.4 mg/mL) overnight at RT, rinsed with PBT, and incubated in anti-rabbit alkaline phosphatase-conjugated secondary antibody at 1:2000 for 1 hour at RT. Following final rinsing, the blot was incubated in NBT/ BCIP (Roche) to produce a blue precipitate. For IP experiments, we used the Seize X IP kit (Pierce Biotechnology, Rockford, IL). The CLRN1-B2 antibody was conjugated to agarose beads by cross linking with cyanogen bromide according to the manufac- turer’s protocol. Approximately 200 mg of total retinal protein (from 4 combined adult mouse retinas) was incubated overnight at 4uC with the agarose bead-bound antibodies. The beads were washed several times, and the immunoprecipitated proteins were eluted 3 times. Ten per cent of each eluate was loaded on each lane of a polyacrylamide gel and processed for standard western blotting (see above). Electroretinography Electroretinography was performed using standard procedures. Briefly, mice and rats were dark adapted overnight, anesthetized with xylazine (13 mg/ml) and ketamine (87 mg/ml) under dim red light illumination, and maintained on a heating pad. Corneas were locally anesthetized with 0.5% proparacaine, and pupils were dilated with 2.5% phenylephrine and 1% atropine sulfate drops. Full field scotopic electroretinograms (ERGs) were acquired following 10 ms flashes of white light delivered by an Espion Colorburst mini-Ganzfeld stimulator (Diagnosys LLC, Littleton, MA). Retinal responses were recorded using gold wire corneal contact lens electrodes. A 6-step stimulation protocol was used with intensities ranging from -4 log candela second per square meter (cd.s/m2) to 1 log cd.s/m2. Three responses were recorded and averaged for each stimulus intensity level. A-wave amplitude was measured from the baseline to the first negative peak. B-wave amplitude was measured from the A-wave negative peak to the most positive peak. WT (2/2) and Clrn1 KO mice aged up to 24 months were subjected to ERG analysis. At least six animals were recorded for each genotype at each age. Data were analyzed using Student’s t-test. Recording traces were averaged for each light intensity level, and error bars represent one standard deviation. Cochlear Immunohistochemistry For whole mount immunohistochemistry, samples were fixed in 4% PFA overnight at 4uC and then washed with PBS. Auditory (from middle and apical turn of cochlea) and vestibular sensory epithelia were isolated and then incubated with 0.05% Triton in 10% normal donkey serum (NDS) for 1 hour at RT. Tissues were incubated overnight with rabbit anti-myosin VI primary antibody (Proteus Biosciences, Ramona, CA) at a dilution of 1:200 in a green buffer (Dyna Scientific, Logan, UT). The tissues were then Clarin 1 Is Necessary for Hearing but Not Vision washed and incubated for 2 hours at RT with a secondary antibody conjugated to AlexaFlour488 and rhodamine-conjugated Phalloidin (Invitrogen - Molecular Probes, Carlsbad, CA), and after final rinsing were mounted with fluorescent mounting medium (GBI-Golden Bridge International, Mukilteo, WA). All images were acquired on a Leica Confocal Microscope TCS-SP5. Cochlear Quantitative Measurement Analysis The length of the stereocilia from the tallest row of the inner hair cells bundle was measured using Image J (http://rsbweb.nih. gov/ij/) and used for quantitative analyses. A minimum of 8 images of inner hair cells were acquired from the mid-apex turn of the cochlea (60–80% of the cochlear length) from three different animals of each genotype. All images were taken with the same magnification and exposure parameters. t-tests were performed using SPSS software. Auditory Brainstem Response Brainstem responses were recorded for Clrn1 KO and WT C57BL/6J mice between P16 and P42. Following deep anesthesia using ketamine and xylazine, animals were maintained on a warming pad, and three needle electrodes (recording, reference, and ground) were inserted subcutaneously. Tone pips (3 ms duration, frequencies at 8, 16, or 32 kilohertz (kHz) and intensities at 40, 60, or 80 dB SPL (decibel sound pressure level)) were delivered to the left ears at a rate of 19 times per second through a calibrated earphone (Stax Ltd., Japan). ABR signals were recorded using BioSigRP software on a Tucker Davis Technology System 3 recording rig (Alachua, FL), and 500 recordings were averaged for each animal in each condition. EGFP-CLRN1 Fusion Construct and Transfection The pEGFP-C1 vector (GI:1377914; Clontech, Mountain View, CA) was utilized for generating a fusion construct between EGFP and CLRN1. The full-length mouse Clrn1 coding sequence (exons 1, 3, and 4, and including a stop codon; 699 bp) was cloned from C57BL/6J genomic DNA. The clone was sequenced and spliced into the BamHI and EcoRI restriction sites within the pEGFP-C1 multiple cloning site at the 39 end of, and in frame with, EGFP. Viable fusion protein expression was evidenced by a redistribution of fluorescence from the cytoplasm to the plasma membrane in transfection assays (see Figure S4). HEK293 cells were used for all transfection assays. Transfection with the EGFP- CLRN1 construct was performed essentially as described previ- ously [72]. Briefly, plasmid DNA was mixed with Lipofectamine 2000 (Invitrogen) and incubated with ,50% confluent HEK293 cells grown on cover glass. After 24–36 hours, the cells were fixed in 10% neutral buffered formalin for 159, and subjected to immunocytochemical analysis. Supporting Information Figure S1 Oligonucleotide primers and fluorescent probes used. Found at: doi:10.1371/journal.pgen.1000607.s001 (0.40 MB PDF) Figure S2 Clrn1 mRNA co-localizes with a marker for Mu¨ ller cells. (A–C) Clrn1 ISH combined with anti-Glast (Slc1a3; a Mu¨ ller glial marker) immunohistochemistry labeling shows co-labeling. The labeling in the GCL represents Mu¨ ller cell processes, which extend to and surround cells in the GCL. (D–F) Higher magnification view of the region in the box in H, and the arrow points to a cluster of cells that is labeled with both Clrn1 and Glast. Scale bars = 50 mm. Found at: doi:10.1371/journal.pgen.1000607.s002 (1.42 MB TIF) PLoS Genetics | www.plosgenetics.org 15 August 2009 | Volume 5 | Issue 8 | e1000607