Immunofluorescent staining

Tca-8113 cells and Ca9-22 cells were grown on precoated coverslips, and fixed in 2% PFA in phosphate-buffered saline (PBS) for 15 min at 37°C. Cells were blocked with 3% BSA in PBS for 30 min at room temperature for non-special staining, and cells were then incubated with primary anti-GFP antibody (1:500) and secondary antibody (Alexa Fluor 488, 1:500) for 1 hr at room temperature orderly. For recording positive fluorescent signals of GFP puncta (green Color), an Olympus FV1000 microscope was used. The fluorescent images were taken with a 60 × oil-immersion lens.