Rhodamine phalloidin dye

Hs578t cells were seeded on a glass coverslip in 6-well plates at 50% of confluence. Following cell attachment the medium was removed and cell were fixed with 4% Paraformaldehyde containing 0.3% Triton X100. Cells were washed three times in 1X PBS and incubated with blocking buffer (0.1% Triton X100, 3% BSA in PBS). Vinculin antibody (Sigma #V4505; dilutiom: 1/50) was used to detect the FAPs. Phospho-Myosin light chain-2 antibody (Cell Signaling Technology #3671) was used at 1/50 dilution. F-Actin was stained with rhodamine phalloidin dye (Invitrogen #R415) at 1/200 dilution. Nuclei were visualized using Hoecht 33342 dye (Invitrogen #H3570) at 10 μg/mL. Images were acquired under a Zeiss 780 confocal microscope.

For immunofluorescence staining, the cells were fixed with 4% paraformaldehyde for 15 min in PBS, permeabilised with 0.2% Triton X-100 in PBS, blocked with 5% BSA in PBS and then stained with primary antibodies, α-tubulin (ab52866, Abcam, Cambridge, MA, USA), α-actinin (A7811, Santa Cruz Biotech, CA, USA) or KIF5B (ab167429, Abcam), followed by Alexa Fluor 546/633 secondary antibodies. Rhodamine phalloidin dye (R415, Invitrogen) and WGA Alexa Fluor® 488 conjugate (WGA, W11261, Invitrogen) were incubated with the cells for 20 min at room temperature for F-actin or cell membrane staining. To detect the mitochondrial transfer shown in Fig. 5a, MitoTracker® Orange CMTMRos (Mito Orange, M7510 Invitrogen) was incubated with the cells for 20 min at 37 °C.
Stained cells were visualised and analysed using a laser scanning confocal microscope with a 63X/1.4NA oil immersion objective lens and excitation wavelengths of 488, 549 and 633 nm. All images were acquired using ZEN 2012 software.

A subset of cochleae were harvested from mice to perform hair cell counts following 60 d of AAE treatment. Mice were deeply anesthetized with isofluorane, decapitated, and the cochleae were dissected and placed in postfix (4% paraformaldehyde). They were decalcified in 5% EDTA for 72 h and dissected under a microscope in 0.01 m PBS. Following dissection of individual quarter to half turns, all sections were stained with a 1:100 dilution of Rhodamine Phalloidin dye (Invitrogen R-415, Thermo Fisher Scientific) for 2 h, and then mounted on drop-slides in glycerol. Scout images were obtained at 50×, and custom software determined the distance from the apex and approximate frequency mapping. Chord lengths were measured at the location of the inner pillar cells on each scout image. Subsequent higher magnification images were then obtained at either 200× or 400× using either fluorescent microscopy or laser scanning microscopy. Hair cells were counted only when the stereocilia pattern could be visualized on the apical surface of the cell. Thirty hair-cell-wide bins (the approximate number visible in a single image at 400×) were counted and the percent missing from each bin were recorded at six separate cochlear regions per exposure group.