Immunofluorescence and Alkaline Phosphatase Staining

For immunofluorescence staining, cells were fixed with 4% paraformaldehyde in PBS for 15 min and blocked by 1% BSA at room temperature for 1 h. Cells were then incubated with primary anti-EpCAM antibody (1:200, ab71916, Abcam) at 4 °C overnight. After washed three times with PBS, cells were incubated with FITC conjugated secondary anti-rabbit antibody for 1 h. Nuclei were stained with 10 ug/mL Hoechst 33342 for 2 min. Images were captured under a Nikon fluorescence microscope. The alkaline phosphatase (AP) staining was determined by AST Fast Red TR and α-Naphthol AS-MX Phosphate (Sigma Aldrich) as our previously described4 (link). Images were captured by a Pentax K-50 digital camera.