Fluorescein isothiocyanate fitc labelled phalloidin

Neuromast hair cells were labelled by exposing live 120hpf larvae to 2.5 μM FM1-43FX for 1 min 30 s in the dark. Larvae were quickly rinsed five times in PBS, and labelled larvae were anesthetized with 3-aminobenzoic acid ethyl ester methane sulphonate (MS-222) and fixed in 4% paraformaldehyde (PFA) overnight at 4°C. Then, DAPI nuclear staining was used to quantify hair cells. Labelled hair cells of neuromasts along the body were recorded on one side of each fish and visualized by confocal microscopy with a 63× oil lens. Cilia in 120hpf zebrafish inner ear were marked with 2.5 mg/ml fluorescein isothiocyanate (FITC)-labelled phalloidin (Sigma) in PBS at room temperature. Light exposure was avoided after fixation in 4% PFA, 2% Triton X-100 (Sigma) in PBS. Finally, embryos were washed several times in PBS for over 2 h and viewed under a confocal microscope.

Human immortalized podocytes were grown on poly-D-lysine-four-well chamber slides to 50–60% confluence. After removing the medium, cells were washed twice thoroughly with PBS followed by fixation with 4% freshly prepared paraformaldehyde and blocking with 5% donkey serum for 20 minutes. The cells were stained with primary anti-synaptopodin (sc-21537, 1 : 200, Santa Cruz Biotechnology Inc., Heidelberg, Germany) and secondary Cy3-conjugated antibodies. For the visualization of the cytoskeleton, fluorescein isothiocyanate- (FITC-) labelled phalloidin (Sigma-Aldrich, St. Louis, MO, USA) at a dose of 50 μg/mL for 40 minutes was applied to stain F-actin. To optimize detection, streptavidin/biotinylated horse-radish peroxidase system was applied for amplification of the signal. Finally, the samples were covered with a DAPI (4′,6-diamidino-2-phenylindole) containing antifade mounting medium (EverBrite mounting medium, Biotium Inc., Hayward, CA, USA) to stain the nuclei and minimize photobleaching of the samples. Pictures were taken using a Leica DM4000 fluorescent microscope equipped with a Leica DFC310 FX digital color camera (Leica Microsystems GmbH, Wetzlar, Germany).