Alexa633 conjugated phalloidin

Immunofluorescence was performed as previously described (Hicks et al., 2015 (link)). In brief, cells were fixed in 4% formaldehyde (Pierce, ThermoFisher) for 5 min at room temperature. The cells were permeabilized by washing in PBST (PBS +0.1% Triton X-100). The primary antibody was incubated in blocking buffer (PBS +5% Normal goat serum) overnight at 4°C. Next, the samples were washed three times in PBST and incubated for 1 h at room temperature with the fluorescent secondary antibody (Goat anti-mouse or Goat anti-rabbit antibodies conjugated with either Alexa488 or Alexa555, 1:400 dilution, Life technologies) in the same blocking buffer. The samples were then washed four times with PBST. In order to visualize nuclei, the cells were stained with DAPI. For visualizing the actin cytoskeleton Alexa633 conjugated Phalloidin was used (Life technologies; 1:400). For detecting biotinylated proteins, the samples were incubated with Streptavidin Alexa647 (Life technologies; 1:1,200). The Strep674 was added to the sample at the same time as the secondary antibody. The cells were mounted onto slides using Prolong Diamond (Life technologies).

Mice were anesthetized, killed and inner-ear tissues were removed. The cochleae were further dissected and fixed in 4% paraformaldehyde”. Immunofluorescence staining with antibody against cleaved Caspase-3 (C-Casp3, rabbit IgG, Promega) was performed on whole-mount preparations of the finely dissected organ of Corti. We incubated the tissues in the antibody solutions for 1 h after blocking. As secondary antibodies, we used Cy3–conjugated anti rabbit IgG (Sigma Aldrich). F-actin was visualized with Alexa 633–conjugated Phalloidin (Life technologies). Fluorescence confocal images were obtained with a LSM510-META confocal microscope (Carl Zeiss). Some of the green fluorescence in Cx26R75W + mice indicated the pseudocolor obtained from the signal of Alexa 633–conjugated secondary antibodies (Invitrogen), because these mice have ubiquitous EGFP expression from their transgene. z-stacks of images were collected at 0.5 μm intervals, and the single image stacks were constructed with LSM Image Browser (Zeiss); three-dimensional images and videos were constructed with IMARIS software (Bitplane). We analyzed at least six samples from three animals, and representative images are shown. The compared images were photographed and processed using identical parameters. Three-dimensional images were constructed with z-stacked confocal images by IMARIS (Bitplane).