Immunofluorescence Analysis of Cell-Cell Junctions

Immunofluorescence studies were performed as previously described [63 (link)]. RBMVEC grown on 12 mm in diameter fibronectin-coated glass coverslips were treated with AMG837 (10 µM, 30 min) and then processed for immunocytochemistry studies; untreated cells served as controls. Cells were first washed with phosphate buffer saline (PBS) and then fixed with 4% paraformaldehyde (20 min, room temperature). Cells were washed in PBS and PBS with 0.5% Triton X for 5 min and incubated in normal goat serum (1:20, 1 h, room temperature). Cells were incubated overnight at 4 °C with the following primary antibodies: ZO-1 (1:200, rabbit polyclonal, Cat # 40–2200, Thermo Fisher Scientific, Waltham, MA, USA) and VE-Cadherin (1:200, rabbit polyclonal, Cat # 36–1900, Thermo Fisher Scientific) followed by incubation with secondary antibody Alexa Fluor 488 goat anti-rabbit IgG (1:200, Cat # A11008, Thermo Fisher Scientific) or Alexa Fluor 568 goat anti-rabbit IgG (1:200, Cat # A11011, Thermo Fisher Scientific) for 2 h at room temperature. Additionally, cells were incubated in ActinRed 555 (Thermo Fisher Scientific) for 30 min at room temperature. Cells were washed in PBS and then mounted with DAPI Fluoromount G (SouthernBiotech, Birmingham, AL, USA) on glass microscope slides. Cells were examined under a Leica DMI6000B fluorescence microscope equipped with the appropriate filters.