Rabbit anti tuj1

The cochlea was fixed in 4% formaldehyde at 4°C overnight and decalcified in 10% EDTA at room temperature for at least 24 h. For sectioning, the cochleae were dehydrated with 15% sucrose for 2 h and later in 30% sucrose overnight at 4°C. Samples were embedded in Tissue-Tek OCT compound and frozen at −20°C and then sectioned into 9-μm-thick slices. For whole-mount immunostaining, the sensory epithelium in the cochlea was isolated and divided into several parts of apical, middle, and basal turns. Then, the sections or whole cochlea samples were permeabilized with 0.5% Triton X-100 in PBS at room temperature for 15 min, washed in PBS thrice, and then blocked in 10% goat serum in PBS at 374°C for 30 min. The samples were incubated with a primary antibody at 4°C overnight. After washing with PBS, further incubation with a secondary antibody (goat anti-rabbit Alexa-488 or Alexa-568, 1:500; Invitrogen) diluted in PBS at 37°C for 1 h was performed, followed by Alexa Fluor 488-conjugated phalloidin (Sigma-Aldrich, United States) at 37°C for 30 min and DAPI at RT for 5 min. Immunofluorescence images were collected using an LSM 880 confocal microscope (Zeiss). The primary antibodies included rabbit anti-LOXL3 (1:200, American Research Products, United States), rabbit anti-myosin VIIa (1:200, Cell Signaling, United States), and rabbit anti-Tuj1 (1:400, Cell Signaling, United States).

Cell samples were lysed in a buffer containing 150 mM NaCl, 10 mM HEPES, 2 mM MgCl₂, 10 mM KCl, 0,5 mM EDTA and 0.5% Triton X-100. Protease inhibitors were added freshly. The Protein concentration was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher) and equal amounts of protein were separated by electrophoresis on SDS/PAGE and blotted onto cellulose membranes (BioTrace NT, Pall). Membranes were blocked with 3% BSA in TBST and then incubated with the primary antibody over night at 4 °C (i.e. sheep anti-Trim71 (R&D Systems) rabbit anti-Ago2 (Cell Signaling) rabbit anti-TuJ1 (Cell Signaling), rabbit anti-Sox2 (Cell Signaling), mouse anti-Tubulin (Sigma).
After washing with TBST, followed by incubation with HRP-conjugated secondary antibodies (Jackson ImmunoResearch), bands were detected by chemiluminescence (Thermo Scientific).

Cells were grown on μ‐slides (Ibidi) and fixed in 4% paraformaldehyde (PFA) in D‐PBS (without Ca²⁺ and Mg²⁺) for 10 min at RT followed by PFA inactivation in 300 mM glycine in D‐PBS (5 min, RT) and a wash in D‐PBS. Cells were permeabilized with 1% (v/v) Triton X‐100, 0.2% (w/v) SDS, 10 mg/ml BSA in D‐PBS (1 h, RT) and incubated with primary antibodies overnight at 4°C in 50 mg/ml BSA in TNT (100 mM Tris–Cl (pH 7.5), 150 mM NaCl, and 0.1% (v/v) Tween‐20). The following antibodies and dilutions were used: rabbit anti‐TUJ1 (Cell Signaling, 5568) at 1:600; rabbit anti‐DESMIN (Cell Signaling, 5332) at 1:300; rabbit anti‐GATA6 (Cell Signaling, 5851) at 1:1,600. An anti‐rabbit IgG (H+L) F(ab′)₂ Fragment Alexa Fluor 647 Conjugate (Cell Signaling, 4414) served as secondary antibody and was allowed to incubate for 2 h at RT in 50 mg/ml BSA in TNT. Nuclei were visualized using a constitutive nuclear marker (a stably integrated CAG::H2B‐mCherry‐BGHpA plasmid). Confocal images were acquired on an inverted SP8 confocal microscope (Leica) equipped with a 40× PL Apo 1.1 W objective. TUJ1 immunostaining for quantification by flow cytometry was performed under the same conditions except that the starting material was a single‐cell suspension.