Shandon sequenza staining rack

Six weeks post infection, the region of the temporal bone containing the cochlea was dissected from skull, fixed in 4% PFA, and then transferred to OsteoSoft® (Merck) for 7–10 days. OsteoSoft was replaced by 30% sucrose in PBS 24 h before cutting. Left cochleas were used for the spiral ganglion histology, whereas the right ones were kept as back-up. Then, 14 μm sections were cut and mounted on Superfrost Plus Menzel glass slides. The staining procedure was performed with a Shandon Sequenza staining rack (Thermo Fisher Scientific). Sections were permeabilized for 5 min with 0.1% Triton-X, followed by blocking with blocking solution (PBS with 2% BSA and 0.01% Triton-X) for 1 h at room temperature (RT). The primary antibody βIII-tubulin (1:500, mouse-anti-rat, Promega G712A) was diluted in blocking solution and incubated overnight at 4 °C. The sections were rinsed with 3 × 500 μl PBS and incubated with the secondary antibody (goat-anti-mouse Alexa Fluor 488, 1:500, Thermo Fisher Scientific, A-11029) for 2 h at RT. After rinsing again, the slides were mounted with Fluoroshield™ with DAPI (Sigma-Aldrich). Images of the spiral ganglions were acquired using a fluorescent microscope (ZEISS Axio, Imager M1, West Germany) equipped with a digital camera (Zeiss AxioCam HRc).

Cochleae were dissected immediately after perfusion of animals and placed for 4 h in 4 % PFA at 4°C and subsequently decalcified with Osteosoft (Merck, Germany) for at least 10 days.
Cochleae were dehydrated stepwise with 15 % for 2 h and 30 % sucrose overnight. Samples were embedded in optimal cutting temperature medium (O.C.T., Sakura, Netherlands) and cut with a cryostat (Leica CM3050 S) in 16 μm thick mid-modiolar sections, where every second section was mounted on Superfrost Plus microscopy slides (Thermo Fisher Scientific, USA) for quantification studies. To stain the samples, slides were put in a Shandon Sequenza staining rack (Thermo Fisher Scientific, USA). Sections were permeabilized for 10 min with 0.1 % Triton X-100 and blocked with blocking solution (2 % BSA, 0.01 % Triton X-100 in PBS) for 1 h at room temperature. Samples were incubated with primary antibodies (Table 1) overnight at 4°C. The next day slides were rinsed and incubated with the corresponding secondary antibody (Table 1) for 2 h at room temperature, rinsed again and mounted with a coverslip using Fluoroshield containing DAPI (Sigma).
Slides were visualized with a Nikon Eclipse Ti-E using a Nikon-PLAN Fluor 4x/0.13 NA, 10x/0.3 NA, 20x/0.50 NA or 40x/1.00 NA Oil objective and by a Zeiss 710 laser scanning microscope using a Zeiss Plan-Apochromat and 40x/1.3 NA oil objective for hair cell quantification.