Alexa 647 phalloidin

Cells cultured on the coverslip were washed with PBS, fixed with 4% paraformaldehyde for 1 h at room temperature and then washed three times with PBS. The cells were blocked with 1% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for 1 h, washed and permeabilized with Cytofix/Cytoperm reagent (Becton Dickinson, Franklin Lakes, NJ, USA) for 10 min at 4 °C and then washed with PBS. To detect viral nucleoproteins (NP), cells were incubated with 1:100 FITC conjugated mouse anti-NP antibodies (Chemicon International) diluted in blocking buffer at room temperature for 1 h. Cells were rinsed with PBS, followed by deionized water, and then mounted in Prolong Gold antifade reagent containing DAPI counterstain (Invitrogen, Foster City, CA, USA). Stained cells were analyzed by fluorescence microscopy (Nikon, Tokyo, Japan). Cells from live-cell imaging were washed with PBS, fixed with 4% paraformaldehyde for 1 h at room temperature. Cells were then blocked with blocking buffer, washed, and permeabilized with 0.1% saponin containing blocking buffer. For F-actin staining, cells were incubated with 1:500 Alexa 647 Phalloidin (Thermo Fisher Scientific, Waltham, MA, USA). Cells were then analyzed by the Operetta HC imaging analysis system (PerkinElmer, Hamburg, Germany).

HEL cells transfected with EGFP-tagged constructs were stimulated with 800 nM PMA for 5 min and incubated with immobilized fibrinogen in duplicate, for 1 h or 2 h at 37 °C in serum-free medium. After extensive washing with PBS, the adherent cells were fixed with 4% PFA and stained antivinculin antibody (hVIN-1 clone, Sigma-Aldrich, St. Louis, MO), followed by secondary antimouse goat F(ab) fragment antibody Alexa 568-conjugated (Thermo Fisher Scientific) and with Alexa 647-phalloidin (Thermo Fisher Scientific). The cells were visualized with 40x and 63X objectives using a Leica TCS-NT laser scanning confocal microscope and the cell area was measured for 150 cells with ImageJ software.
HeLa cells after CRISPR or cell sorting were seeded to fibronectin-coated coverslips (10 µg/cm²) for 2 h spreading at 37 °C. Adherent cells were fixed with 4% PFA and stained with anti-vinculin antibody (Sigma) or anti-GFP antibody (Abcam) and anti-ILK antibody (abcam) followed by goat anti-mouse antibody Alexa 488-conjugated (abcam) or goat anti-chicken antibody Alexa 488-conjugated (Abcam) and goat anti-rabbit Alexa 568-conjugated (abcam). Coverslips were then mounted with Prolong Gold Antifade Reagent with DAPI (Fisher) overnight and visualized with Leica TCS-SP5 II upright confocal microscope (Leica Microsystems, GmbH, Wetzlar, Germany).

NRK cells with or without Lifeact-mGFP expression were fixed with 4% paraformaldehyde in PBS at room temperature for 1 h, and washed three times with PBS. After an incubation with 0.1% Triton X-100 for 5 min and blocking with 5% skim milk for 1 h, the cells were stained with 500 nM Alexa647-phalloidin (Thermo Fisher Scientific) for 1 h, washed with PBS, and mounted in Permafluor medium (Thermo Fisher Scientific). Observations were performed with a 100×, 1.4 NA objective lens at room temperature using an Olympus FV-OSR system, relying on the reduced pinhole size and the software to enhance the high spatial frequency components. The pixel size of the final images is 43 nm.

Immunocytochemistry was employed to probe the abundance of cytochrome c oxidase and catalase in control and KA-treated cochlear explants, as well as the cellular localization of BDNF and TrkB in cryostat sections of untreated p3 mouse cochleae. The primary antibodies (Table S1) used were anti-cytochrome c oxidase (1:200, BD Biosciences, #556432 RRID:AB_396416), anti-BDNF (1:500 Abcam, Amsterdam, Netherlands, #ab223354), and anti-TrkB (1:500, Abcam, #ab18987 RRID:AB_444716). The samples were counterstained with Alexa 647 phalloidin (1:1000, Thermo Fisher Scientific, #A22287) to visualize filamentous actin and Hoechst 33342 (1:5000, Thermo Fisher Scientific group Illkirch, France, #62249) to stain the nuclei. The secondary antibodies used were as follows: donkey anti-mouse and anti-rabbit IgG conjugated to Alexa 488 (Molecular Probes, Illkirch, France, #A-21202 RRID: AB-141607). Fluorescent tags were visualized using confocal microscopy (Zeiss 880 Airyscan, Zeiss group, Germany). In control samples without primary antibodies, Alexa 488 fluorescent tags were not observed. Immunocytochemistry assessments required 3 to 5 cochleae per condition.

For immunocytochemistry, BMMCs (1 × 10⁶) were grown on sterilized round cover glasses (Matsunami Glass, Osaka, Japan) with M-CSF and RANKL in a 24-well plate. BMMCs were fixed with 3.7% formaldehyde for 10 min, blocked with 1% BSA in PBS for 1 h at room temperature and then incubated with anti-Sema4D mouse monoclonal antibody conjugated with FITC (BD) anti-PlexinB2 mouse monoclonal antibody conjugated with PE (BD), anti-CD72 mouse monoclonal antibody conjugated with PE (BD Biosciences), Alexa 647 Phalloidin (Thermo Fisher Scientific) and DAPI (BD Biosciences) overnight at 4 °C. The next day, cells were embedded in Fluoromount-g (Thermo Fisher Scientific). Immunofluorescence signals were observed using the Zeiss LSM780 Confocal Microscope (Carl Zeiss, Dublin, CA, USA).

MDCK cells, 72 h post transfection, were rinsed with PBS, fixed in 4% paraformaldehyde at room temperature for 20 min and blocked with 10% HS in 0.1% Triton X-100/PBS. The cells were then incubated with primary antibody (1,1000 anti-Myc, Biolegend; 1,1000 anti-HA, Biolegend) with 3% HS in 0.1% Triton X-100/PBS, overnight at 4 °C. The following day, the cells were washed in 0.1% Triton X-100/PBS and incubated, for 1 h at room temperature, with the Alexa-546 and Alexa-488 conjugated secondary antibodies (Life Technologies) with 3% HS in 0.1% Triton X-100/PBS. Filamentous actin was stained with Alexa-647 phalloidin (ThermoFisher) in 0.1% Triton X-100/PBS for 1 h. The cells were then washed and the coverslips were mounted on glass slides, using FluoroGel mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA).
All images were acquired using a Zeiss LSM880 Fast Airyscan scanning confocal microscope equipped with 63x objective, and the analyses were performed using ImageJ software.