4 well tissue culture chambers

Cells were treated and grown in sterile 4-well tissue culture chambers (Sarstedt, Nümbrecht, Germany). They were washed with PBS once before fixed with 4% PFA for 15 min at room temperature, then washed with PBS for 3 times and permeabilized in 0.1% Triton X-100/PBS for 15 min. The slides were blocked with 5% goat serum in 0.1% Triton X-100/PBS for 1 hour at room temperature. Actin was stained with eflour660-phalloidin (1:200, Invitrogen) for 1 hour and with SYTOXGreen dye (1:5000, Invitrogen) for nuclei staining for 30 min in the dark. The slides were mounted with ProLong Gold antifade reagent (Invitrogen). Confocal microscopy was performed with a confocal laser-scanning microscope (LSM 5 Exciter, Carl Zeiss, Oberkochen, Germany) with a C-Apochromat 63/1.3 NA DIC water immersion objective. The mean fluorescence from six related cells of each picture was quantified by ZEN software (Carl Zeiss).

Cells were treated and grown in sterile 4-well tissue culture chambers (Sarstedt,Germany). They were washed with ice-cold PBS once and then fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature. Cells were washed 3x with ice-cold PBS, and permeabilised with 1x permeabilization buffer (eBioscience) for 15 minutes at room temperature. E-Cadherin primary antibody (#3195; Cell Signaling) [diluted in permeabilization buffer (1:100)] was added and kept in a humidified chamber at 4°C overnight. The following day, cells were washed with ice-cold PBS (x4) and incubated with the secondary antibody goat anti-rabbit IgG-FITC (#sc-2012; Santa Cruz Biotechnology) (1:200) for 1hour at room temperature. The chambers were then washed three times with ice-cold PBS. Then DRAQ5™ (nuclear stain; eBioscience) was added. To mount Ishikawa cells CC/Mount (Sigma) was added and the cover slip was placed. Confocal microscopy was performed with a confocal laser-scanning microscope (LSM 5 Exciter, Carl Zeiss, Germany) with a C-Apochromat 63/1.3 NA DIC water immersion objective. (Carl Zeiss, Germany)