2.7 Immunocytochemistry
Human or rat DP cells were seeded in 12-well plate on cover slips. The cells were incubated with KY19382 for 48 h. Cultured cells were washed twice with cold PBS and were fixed in 4% paraformaldehyde or 10% formalin for 15 min at room temperature, and then were washed with PBS and permeabilized with 0.2% Triton X-100 for 15 min. After blocking with 5% BSA in PBS for 30 min at room temperature, the cells were blotted with primary antibody: β-catenin antibody (1:100, BD Transduction Laboratory, Lexington, KY or 1:50, Abcam) overnight at 4°C. After washing with PBS, the cells were blotted with Alexa Fluor 488-conjugated goat anti-mouse antibody or Alexa Fluor 555-conjugated goat anti-rabbit antibody (1:300, Molecular Probes, Leiden, The Netherlands) for 1 h at room temperature and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:5000, Boehringer Mannheim, Mannheim, Germany) for 10 min at room temperature. Images were taken using a LSM510 confocal microscope (Carl Zeiss Inc., Thornwood, NY). The fluorescence intensity was quantified using NIS-Elements AR 3.2 software (Nikon).