4 6 diamidino 2 phenylindole dapi staining

To visualize HCs, OC explants were subjected to FITC-phalloidin staining. Subsequently, the explants were cultured for 24 h and fixed in 4% paraformaldehyde (PFA; Electron Microscopy Sciences, Hatfield, PA, USA; Bas et al., 2012 (link)). Next, the explants were washed three times in phosphate-buffered saline (PBS; Sigma–Aldrich, St. Louis, MO, USA) and incubated in 5% normal goat serum (Sigma–Aldrich, St. Louis, MO, USA) and 1% Triton X-100 (Fluka, St. Louis, MO, USA) in PBS for 90 min at 25°C. Then the samples were washed three times with PBS and incubated with FITC-labeled phalloidin (Sigma–Aldrich, St. Louis, MO, USA) for 90 min at 25°C. Additional three washes were performed with PBS and then the samples were mounted with mounting medium having 4′,6-diamidino-2-phenylindole (DAPI) staining (Vector Laboratories, Burlingame, CA, USA), coverslipped, and viewed under a confocal Zeiss Axiovert 700 microscope (Carl Zeiss Microimaging, LLC, Thornwood, New York, NY, USA).

In parallel, AM pieces were used for assessing cell viability at each time point. To this aim, the vital quantifiable fluorescent Calcein AM (C1359 Sigma-Aldrich), propidium iodide (P4864 Sigma-Aldrich), and 4′,6-diamidino-2-phenylindole (DAPI) staining (Vectastain) were used. Briefly, working in the dark, Calcein AM, DAPI, and propidium iodide were consecutively added to the culture medium (45, 5, and 2 min, respectively, before performing the subsequent analysis) according to the manufacturer's instructions. For the fluorescence analysis, Nikon A1r confocal microscope interfaced to a computer workstation, provided with NIS-Elements 4.4 software (for images acquisition) and with NIS-Elements Advanced Research imaging software (for post-processing analysis), was used. Quantification of Calcein AM, propidium iodide, and DAPI fluorescence was performed by using the image-processing software ImageJ, version 1.40g (http://rsbweb.nih.gov/ij). The viability of the fresh AM was taken as 100%. Each experimental group was analyzed in triplicate, and the relative levels of fluorescence were obtained from 10 random fields.