Radiance 2000 laser scanning confocal microscope

Cells grown on glass coverslips were fixed with ice-cold 4% paraformaldehyde for 15 min, followed by permeabilization with 0.2% Triton X-100 and incubation with normal goat serum for 30 min at 37°C. Cells were then incubated overnight with p120ctn mouse monoclonal antibody (1∶200, cat. 610134; BD Transduction Laboratories, Lexington, KY, USA) and E-cadherin rabbit polyclonal antibody (1∶100, SC-7870; Santa Cruz Biotechnology). Primary antibodies were applied overnight at 4°C, followed by incubation with a rhodamine/fluorescein-5-isothiocyanate (FITC)-labeled secondary antibody goat anti-mouse or TRITC-labeled goat anti-rabbit IgG (1∶100, cat. E031210-01 and E031320-01, EarthOx, San Francisco, CA, USA). The nuclei were counterstained with propidium iodide/4, 6 diamidino-2-phenylin-dole. Epifluorescent microscopy was performed using an inverted Nikon TE300 microscope (Melville, NY, USA), and confocal microscopy was performed using a Radiance 2000 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY, USA).

The Cells were seeded in glass coverslips and fixed with 4% paraformaldehyde in phosphate-buffered saline for 20 min and then permeabilized with 0.2% Triton X-100 for 15 min at room temperature. After washing with phosphate-buffered saline, cells were blocked with normal goat serum for 1 h at room temperature, and then incubated with primary antibody overnight at 4°C. Cells were then incubated with the second antibodies (Dylight488 labeled goat anti-rabbit, Dyligh594 labeled goat anti-rabbit, Dyligh594 labeled goat anti-mouse) and mounted with DAPI. Images were acquired by using a Radiance 2000 laser scanning confocal microscope (Carl Zeiss, Thornwood, NY, USA).

Cells were fixed with 4% paraformaldehyde, blocked with 5% BSA, and incubated with anti-Flag (1:1600, #8146, CST) and anti-β-catenin antibodies (1:100, #8480, CST) overnight at 4 °C, followed by incubation with anti-rabbit IgG (Alexa Fluor 488 Conjugate) (1:1000, #4412, CST) and anti-mouse IgG (Alexa Fluor 594 Conjugate) (1:1000, #8890, CST) at room temperature for 1 h. Cells were counterstained with 4’,6-diamidino-2-phenylindole (DAPI). Confocal microscopy was performed using a Radiance 2000 laser scanning confocal microscope (Carl Zeiss, Germany).