Nuclear fast red

We used the inserted β-galactosidase/LacZ reporter gene to indicate the location of expression of each gene, as described previously (Buniello et al., 2016 (link); Chen et al., 2014 ). Inner ears were dissected from postnatal day (P)14 mice and fixed in 4% paraformaldehyde for 30–40 min with rotation at room temperature. Samples were washed with PBS twice for 20 min and decalcified in EDTA at 4 °C over 72 h. The inner ears were washed in detergent wash (2 mM MgCl₂; 0.02% NP-40; 0.01% sodium deoxycholate in PBS, pH7.3) for 30 min at room temperature. X-gal (Promega, cat. no. V394A) was added 1:50 to pre-warmed staining solution (5 mM K₃Fe(CN)₆ and 5 mM K₄Fe(CN)₆ in detergent solution), and the samples were then incubated at 37 °C until each sample was deemed adequately stained. Following X-gal staining, the samples were washed with PBS, dehydrated and embedded in paraffin wax. The samples were sectioned at 8  μm, counterstained using Nuclear Fast Red (VWR, cat. no. 342094W) and mounted using Eukitt quick-hardening mounting medium (Sigma-Aldrich) before being viewed and photographed using a Zeiss Axioskop microscope connected to an AxioCam camera and interfaced with Axiovision 3.0 software.

To verify internalization of unlabeled nanoparticles, cells were stained with Prussian Blue and counterstained with nuclear fast red. Briefly, cells were fixed with 4% paraformaldehyde (PFA; AppliChem, Darmstadt, Germany) for 10 min at room temperature (RT) and washed twice with phosphate buffered saline (PBS; Biochrom, Berlin, Germany). Cells were incubated for 20 min in a mixture of 1% HCl (Carl Roth, Karlsruhe, Germany) and 2% potassium ferrocyanide (AppliChem) at equal volumes, washed twice with PBS and counterstained using 1% nuclear fast red (VWR International GmbH) in 5% aqueous aluminium sulfate (AppliChem) solution for 20 min. Cells were washed twice with PBS and once with distilled water. Finally, cells were embedded in mounting medium consisting of 12% poly(vinyl alcohol) (w/v; Sigma-Aldrich Chemie GmbH), 30% glycerol anhydrous (w/v; AppliChem), 0.53 mM phenol (Sigma-Aldrich Chemie GmbH) and 60 mM TRIS (pH 8.5; AppliChem). After polymerization of mounting medium, stained cells were investigated using the microscope Zeiss Axiovert 40 CFL (Carl Zeiss Microscopy GmbH, Jena, Germany).

Heads from mice aged 4 weeks were bisected through the midline, the brain removed, and semicircular canal opened, then fixed in 4% paraformaldehyde at RT for 90 minutes. After decalcification in EDTA, the samples were washed for 30 minutes at RT with rotation with the detergent solution (2 mM MgCl₂; 0.02% NP-40; 0.01% sodium deoxycholate; 0.01% sodium deoxycholate in PBS, pH 7.3). X-gal (Promega, cat. no. V394A) was added 1:50 to prewarmed staining solution (5 mM K₃Fe(CN)₆ and 5 mM K₄Fe(CN)₆ in detergent solution), and then the samples were stained at 37°C in the dark overnight. The half-skulls were then washed twice with saline at 4°C in rotation for at least 2 hours and stored at 4°C in 70% ethanol until tissue processing and embedding. The samples were gradually dehydrated in ethanol (Leica TP1020 tissue processor) and embedded in paraffin using xylene as clearing agent (Leica EG1150H tissue embedder). The samples were cut at 8 μm thick, counterstained with Nuclear Fast Red (VWR, cat. no. 342094W), and mounted with Eukitt mounting medium (Sigma-Aldrich). Specimens were imaged using a Zeiss Axioskop microscope connected to an AxioCam camera and interfaced with Axiovision 3.0 software.