N used 1:10 (tissue culture supernatant) 1:501:Cadherin 23 (chicken)Cadherin 23 (mouse)Recombinant fragment (aa 437781) of the predicted chicken cadherin 23 sequence (XP_421595) fused to a polyhistidine tag Peptide sequences derived from ectodomain of mouse cadherin 23 (NH2CRGPRPLDRERNSSHCOOH and NH2GDISVLSSLDREKKDHCOOH derived from exons 29 and 52 respectively) conjugated to keyhole limpet hemocyanin1:LSM510 confocal microscope working with a 100oil immersion lens NA 1.four. Tissues from the waltzer v2J pups have been kindly provided and genotyped by Jennifer Hilton and Prof. Karen Steel (Wellcome Trust Sanger Institute, Cambridge, UK).ImmunolabelingAvian inner ear tissues have been obtained from 2dayold chicks. Inner ears have been dissected in PBS pH 7.2, fixed in four paraformaldehyde buffered with 0.1 M sodium phosphate buffer pH 7.four for 1 hours at space temperature, and washed in PBS. Otoconial membranes with adherent otoconia had been removed from utricular maculae with fine forceps prior to fixation; tectorial membranes were removed from the basilar papillae just after fixation. Washed tissue pieces were incubated in preblock (TBS/HS) for 1 hour and then in preblock containing 2 mM EDTA plus a mixture of mAb G19 and R805 overnight. Soon after washing in TBS, tissues had been labeled with either fluorescent or gold conjugated secondary antibodies. In some experiments staining was performed inside the absence of EDTA. For confocal microscopy, tissues were labeled with a mixture of AlexaFluor 488 goat antimouse and AlexaFluor 555 donkey antirabbit IgG, each at a dilution of 1:500 in preblock containing 0.1 TX100 and AlexaFluor 350 phalloidin. For immunogold transmission electron microscopy, tissues had been labeled having a mixture of five nm gold antirabbit IgG and 10 nm gold antimouse IgG. For immunogold scanning electron microscopy tissues had been labeled using a mixture of 20 nm gold antirabbit IgG and ten nm gold antimouse IgG. Fluorescently labeled tissues had been mounted in Vectashield and viewed having a Zeiss LSM510 confocal microscope making use of a 100Planapochromat objective, NA 1.4. For transmission electron microscopy, goldlabeled tissues had been washed, refixed in 2.5 glutaraldehyde in 0.1 M cacodylate buffer pH 7.two containing 1 tannic acid,washed in buffer, and postfixed in 1 osmium tetroxide. Following a short wash with H2O, samples were dehydrated via rising concentrations of ethanol and imbedded in TAAB 812 resin. Thin sections had been reduce having a diamond knife, mounted on copper mesh grids, double stained with uranyl acetate and lead citrate, and viewed within a Hitachi 7100 electron microscope operating at one hundred kV. Images had been captured with a Gatan camera at 2048 2048 pixel resolution. For scanning electron microscopy, goldlabeled tissues have been washed, refixed in two.5 glutaraldehyde, osmicated, dehydrated with ethanol, and vital pointdried from liquid CO2. Immediately after rotary evaporative carbon Fomesafen Protocol coating, the tissue samples were examined within a field emission Jeol 6700F SEM using secondary and backscatter electron detectors. For standard transmission electron microscopy, tissues had been ready as described previously (Goodyear and Richardson, 1992). Figures were 5-Methoxysalicylic acid Cancer constructed employing Adobe Photoshop CS4 (San Jose, CA) and minor adjustments to image contrast and brightness were made to some images.Results Properties with the protocadherin 15 and cadherin 23 antibodiesMany antibodies to cadherin 23 and protocadherin 15, in particular these raised to peptides or recombinant fragments, only stain hair bundles.