Eclipse te2000 e system

Primary cells were seeded at 1.5 × 10⁵ cells/well on glass coverslips in 6-well plates and fixed in ice-cold methanol (5 minutes at −20°C) or 2% paraformaldehyde (20 minutes at room temperature). Permeabilization, blocking methods, and immunofluorescence (IF) staining were essentially as described previously.31 (link) Primary antibodies were used at final dilutions of ×200 to 1000. Secondary antibodies were diluted ×500. Tissues were permeabilized and blocked (0.1% Triton-X 100 with 10% normal goat serum in PBS) for 30 minutes at room temperature and then incubated in primary antibodies overnight at 4°C. After several PBS washes, they were incubated with secondary antibodies for 30 minutes at room temperature. Samples were mounted on glass slides using Vectashield with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Ltd, Peterborough, United Kingdom). Imaging was carried out using a Nikon Eclipse TE2000-E system, controlled and processed by EZ-C1 3.50 (Nikon UK Ltd, Kingston-upon-Thames, United Kingdom) software.

Cells were seeded at 1.5 × 10⁵ cells/well on glass coverslips in six-well plates and fixed in ice-cold methanol (5 minutes at −20 °C) or 2% paraformaldehyde (20 minutes at room temperature). Permeabilization, blocking methods and immunofluorescence staining were essentially as described previously^{28 (link)}. Primary antibodies were used at final dilutions of x200-1000. Secondary antibodies were diluted x500. Confocal images were obtained using a Nikon Eclipse TE2000-E system, controlled and processed by EZ-C1 3.50 (Nikon Inc.) software. Images were assembled using Adobe Illustrator CS4.

For TMEM67 immunofluorescence experiments cochleae were fixed using 2% PFA in phosphate-buffered saline (PBS) for 20 min at room temperature. For morphogenesis studies cochleae were fixed using 4% PFA in PBS overnight at 4°C. The organs of Corti were dissected, and divided lengthwise two or three times for subsequent mounting. Tissues were permeabilised and blocked (0.1% Triton-X 100 with 10% normal goat serum in PBS) for 30 min at room temperature, and then incubated in primary antibodies overnight at 4°C. Following several washes with PBS, tissues were incubated with secondary antibodies (Alexa-Fluor-568-conjugated goat anti-mouse IgG, Alexa-Fluor-488-conjugated goat anti-rabbit IgG and Alexa-Fluor-488-conjugated goat anti-mouse IgG; Life Technologies Ltd) in the dark for 30 min at room temperature. Cells or tissues were mounted on glass slides using Vectashield with diamidino-2-phenylindole (DAPI; Vector Laboratories Ltd, Peterborough, UK). Imaging was carried out using a laser scanning confocal microscope (LSM510; Carl Zeiss Microscopy GmbH, Jena, Germany) or a Nikon Eclipse TE2000-E system, controlled and processed by EZ-C1 3.50 (Nikon UK Ltd, Kingston-upon-Thames, UK) software. Images were assembled using Adobe Illustrator CS4 (Adobe Systems Inc., San Jose, CA, USA).