Immunocytochemical Characterization of Primary BECs

Primary BECs were characterised by positive staining of E-Cadherin (Abcam 15148, Abcam, Cambridge, UK), pan-cytokeratin (sc-8018, Santa Cruz Bio technology, Santa Cruz, CA, USA), or cytokeratin-14 (Abcam 9220) and by negative staining for fibronectin (Abcam 23751) as described earlier [19 (link)]. In brief, cells were grown on 8-well chamber slides (Sarstedt, Sevelen, Switzerland) until 80% confluence and fixed for 2 × 5 minutes in 4% formalin. Cells were permeabilised for 5 minutes in PBS containing 0.1% TWEEN-20 and 0.05% Triton-X100 before being washed 3x with PBS-T (PBS + 0.1% Tween-20), and unspecific binding was blocked by 30-minute incubation in 2% skim milk in PBS-T and overnight incubation with one primary antibody. The slides were then washed 3x with PBS and incubated for 30 minutes at room temperature with a fluorescence labelled antibody specific for the species of the primary antibody (anti-mouse cat# R37114, anti-rabbit cat# R37119, Thermo Fisher). Slides were washed 3x with PBS before being monitored for fluorescence on an EVOS live-cell imaging station (Thermo Fisher).