At the first and last days of in vitro culture, the cells or EBs were sampled for immunostaining. The samples were washed with PBS and immediately fixed with cold 4% paraformaldehyde. The samples were treated with 0.25%
Triton X-100 (Sigma-Aldrich) in PBS for 10 min at room temperature (RT) for permeabilization, followed by 10% normal goat serum (NGS; Vector Laboratories, Burlington, ON, Canada) to reduce nonspecific antibody binding. Morphological changes and expression of hair cell markers for conditioned HEI-OC1 cells and co-cultures were observed through immunofluorescence staining and confocal microscopy (
FW3000; Olympus, Tokyo, Japan). Primary antibodies used were Myo7a and Sox2 (1:200; Millipore, Burlington, MA, US);
E-Cadherin (E-Cad, 1:250) and
N-Cadherin (N-Cad, 1:100; BD Biosciences, San Jose, CA, US); Pax2, Pax8 and
Epcam (1:100; Abcam, Cambridge, UK); Brn3c (1:25) and
Fbxo2 (1:50; Santa Cruz Biotechnology, Dallas, TX, US); and Anxa4 (1:50; R&D Biosystems, Minneapolis, MN, US). Samples were all incubated overnight at 4 °C followed by incubation with corresponding Alexa Fluor 568 or 594 conjugated secondary antibody (1:1000; Abcam) for 1 h at RT. Cells were also stained with FITC-conjugated phalloidin (F-actin) and 4′,6-diamidino-2-phenylindole (DAPI, nuclei).
Carpena N.T., Chang S.Y., Abueva C.D., Jung J.Y, & Lee M.Y. (2021). Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells. Scientific Reports, 11, 13893.
