Immunofluorescence Staining of Cell-Cell Junctions

For immunofluorescence, cells were fixed and permeabilized either with methanol-acetone or, in case of phalloidin staining, with 3% formaldehyde–1% Triton X-100, or, in case of anti-vinculin staining, with BM[PEO]3 (see Indra et al., 2013 (link) for details). Wide-field images were taken using a microscope (Plan Apochromat 100×/1.40 NA objective lens; Eclipse 80i; Nikon) and a digital camera (CoolSNAP EZ; Photometrics). The images were then processed using NIS-Elements software (Nikon). The following antibodies were used: mouse anti–E-cadherin and anti-occludin (Invitrogen), rabbit anti-Dendra2 (Evrogen), mouse anti–β-catenin and ZO1 (BD), mouse anti-vinculin and rabbit anti-EPLIN (Sigma-Aldrich), goat anti–α-catenin (Santa Cruz Biotechnology, Inc.), rabbit anti–α-catenin N-terminal domain, EP1993Y (Abcam), and guinea pig anti-cingulin (provided by I. Hofmann, German Cancer Research Center, Heidelberg, Germany). Alexa Fluor 555 phalloidin and Latrunculin A were purchased from Invitrogen.