Leica sp5 confocal fluorescence microscope

After fixation, cochlear samples were blocked with 10% normal donkey serum in 10 mM phosphate-buffered saline (PBS, pH 7.4) with 0.3% Triton X-100 for 1 h at room temperature and then incubated with primary antibody overnight at 4 °C. The next day, the tissues were incubated for 2 h at 4 °C with 488- or 594-conjugated donkey secondary antibody (Invitrogen) and DAPI (Sigma-Aldrich). Omission of primary antibody served as the negative control. The following primary antibodies were used: anti-β-catenin (BD Biosciences), anti-myosin VIIA (Myosin7a) (Proteus Biosciences, Ramona, CA, USA), anti-cleaved caspase-3 (Cell Signaling Technology), anti-Foxo3 (Cell Signaling Technology), anti-Parvalbumin (Sigma-Aldrich), TUNEL (Roche, Indianapolis, IN, USA), and MitoSOX Red (Life Technologies, Rockford, IL, USA). Cochleae were dissected into apical, middle, and basal turns, and images were taken using a Leica SP5 confocal fluorescence microscope (Leica Microsystems, Biberach, Germany).

hCMEC/D3-MDR1-EGFP cells were plated on collagen type I (100 µg/ml; Invitrogen)-coated 100-mm tissue culture plates (Sarstedt) containing a 42-mm glass plate (H. Sauer Laborbedarf, Reutlingen, Germany). Four days after reaching confluence cells were pretreated with MMC (1 µM) for either 1 or 3 h in Opti-MEM (Invitrogen) at 37°C and 5% CO₂. Cells were stained for 0.5 h with 5 mM bisbenzimide H (Sigma-Aldrich) in Opti-MEM (Invitrogen) at 37°C and 5% CO₂. For confocal fluorescence microscopy glass plates with cells were fitted into a PeCon open chamber (PeCon, Erbach, Germany) and MMC (1 µM) in Opti-MEM without phenol red (Invitrogen) was added. Fluorescence images were taken every 3.6 min for the next hour using a Leica SP5 confocal fluorescence microscope with a 63× water objective (Leica Microsystems, Bensheim, Germany) in a climate box (PeCon) at 37°C. Excitation wavelengths of 405 nm (bisbenzimide H) or 481 nm (Pgp-EGFP) were used.

E. coli BL21 cells expressing ZapB-GFP and ZapB-mCherry proteins at 30 °C for 12 h were centrifuged and resuspended in PBS pH 7.4 to an OD of 0.1. 10 µL of resuspended cells were deposited on top of microscopy poly-l-lysine glass slides, covered with coverslips and observed in a Leica SP5 confocal fluorescence microscope (Leica Microsystems, Germany).