Fig. 6: Anti-HA staining of HA-tagged mini-PCDH15-V4 demonstrates localization at the lower end of the tip link.
From: Mini-PCDH15 gene therapy rescues hearing in a mouse model of Usher syndrome type 1F
a Representative confocal microscopy images at P35 from apical, middle and basal turns of the cochlea show anti-HA staining of HA-mini-PCDH15-V4 (magenta) at the tips of stereocilia (co-stained for phalloidin, green) in knockout cochleas treated at P1 with AAV-HA-mini-PCDH15-V4. IHCs inner hair cells, OHCs outer hair cells. b Transduction efficiency in IHCs and OHCs at P35 in treated Pcdh15fl/fl,Myo15-Cre+/− conditional knockout mice (n = 12). Data are presented as mean values ± SEM. c Quantification of fluorescence intensity of anti-HA labeling in IHCs and OHCs from apical, middle and basal turns of the cochleas in mice treated with AAV-HA-mini-PCDH15-V4. Symbols represent integrated fluorescence intensity in arbitrary units (arb. units) in individual cells (apex: n = 56 IHCs, n = 144 OHCs; middle: n = 49 IHCs, n = 198 OHCs; base: n = 42 IHCs, n = 138 OHCs). Cells were measured from four separate cochleas. Data are presented as mean values ± SD. Significance was determined by a two-tailed unpaired t-test. P values: apex IHCs vs. middle IHCs, p = 0.45 (ns); apex IHCs vs. base IHCs, p < 0.0001 (****); middle IHCs vs. base IHCs, p < 0.0001 (****); apex OHCs vs. middle OHCs, p = 0.40 (ns); apex OHCs vs. base OHCs, p < 0.0001 (****); apex OHCs vs. base OHCs, p < 0.0001 (****). d Immunogold scanning electron microscopy demonstrated localization of HA-tagged mini-PCDH15-V4 at the tips of stereocilia (yellow arrow), similar to the localization of wild-type PCDH15 detected with anti-PCDH15, confirming that mini-PCDH15-V4 targets properly. Scale bars: (a) 5 μm, (d) 100 nm. Source data are provided as a Source Data file.
