Immunofluorescence Staining of GCPs

The tissue samples were stained with anti-LacZ, anti-caspase 3, or anti-Slc7a11 as described elsewhere^{67 (link),68 (link)}. GCPs were fixed in 4% formaldehyde for 10 min, washed with PBS, incubated with a blocking solution containing 10% fetal bovine serum and 0.1% Triton X-100 in PBS for 15 min to block non-specific binding sites, and incubated overnight with the anti-Atoh1 (1:400; Chemicon; AB5692), anti-LacZ antibody, or anti-Slc7a11 primary antibodies. After washing with PBS-Tween, the cells were incubated for 1.5 h with Alexa Fluor594-labeled goat anti-rabbit IgG (Life Technologies, Grand Island, NY, USA; A11037) and processed using 4′,6-diamino-2-phenylindole to visualize cell nuclei (1:3000; 5 mg/ml stock in dimethyl sulfoxide, Merck). Cells were mounted on slides using ProLong Gold Antifade Mountant (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA; P36934), and cell immunofluorescence was visualized using an Olympus FV1000 confocal laser scanning microscope.