Journal: Frontiers in Molecular Neuroscience

Article Title: Overloaded Adeno-Associated Virus as a Novel Gene Therapeutic Tool for Otoferlin-Related Deafness

doi: 10.3389/fnmol.2020.600051

Figure Lengend Snippet: Tonotopic analysis of fl-Otof transduction rates. (A) Representative 3D maximum projection of a lightsheet fluorescence microscopy data set of an Otof- KO mouse cochlea (p24, injected with PHP.eB-CMV/HBA-fl-Otof at p6). For context marking, IHCs and SGNs—including the afferent auditory nerve fibers—were stained against the cytosolic calcium buffer calretinin. (A') Tonotopic mapping of the masked IHC row based on Greenwood's function (Greenwood, ). Parameters were set according to Müller et al. resulting in a frequency range from a minimum of 5.1 kHz to a maximum of 83.1 kHz. The blue dotted line indicates reference points for the frequency-mapping algorithm in which a total of 500 coordinates were interpolated to fit the cochlear spiral. Selected frequency labels indicating the projected boundaries of the defined frequency bands (white dots) are indicated in the figure panel. Please note that the 4–8 kHz region was structurally less well-preserved in several of the processed cochleae, thus preventing meaningful analysis of this particular frequency band using this approach. (B) Representative maximum projections of the indicated frequency regions at higher magnification. IHCs were immunohistochemically-labeled against calretinin and otoferlin to assess viral transduction rates. (C) Quantification of IHC transduction rates in the indicated frequency regions using this approach: mean ± SEM and estimate from individual mice (blue spheres). Scale bars. (A) 200 μm; (B) 50 μm.

Article Snippet: Then, primary antibodies against Calretinin (Cat.-Nr. 214 106; Synaptic Systems) and Otoferlin (Cat.-Nr. 178 003; Synaptic Systems) were diluted in blocking solution and samples were incubated in the primary antibody solution for 48 h. After primary incubation, samples were washed in PBS for 24 h. The secondary antibodies goat anti-chicken 488 (Thermo Fisher, #A-11039) and goat anti-rabbit 647 (Thermo Fisher, #A-21244) were diluted in blocking solution and applied for 48 h. Finally, specimen were washed for 48 h and prepared for tissue clearing according to the iDISCO+ protocol (Renier et al., ).

Techniques: Transduction, Fluorescence, Microscopy, Injection, Staining, Labeling