For immunofluorescence analyses, cells were fixed in 3% paraformaldehyde/3% sucrose and permeabilised with 0.2% Triton X-100. After staining, the coverslips were mounted in ProLong® Gold antifade reagent (Invitrogen) or directly in PBS (for TIRF microscopy). Wide-field fluorescence was observed through 40X/1.3 oil objectives on a Zeiss Axiovert 200M microscope equipped with a CoolSnap HQ CCD. Image acquisition was performed using the MetaMorph Imaging System. Confocal imaging was performed on a Zeiss NLO780 confocal system using a 63X/1.4 objective. Total Internal Reflection Fluorescence (TIRF) microscopy was performed on our prototype system including 100x/1.49O Nikon objective, Ixon3 EMCCD (Andor, Belfast, Northern Ireland) and a laser bench with 491 nm (Oxxius, Lannion, France) and 561 nm (Cobolt, Solna, Sweden).
Images were analysed using MetaMorph Imaging System and Volocity (PerkinElmer, Waltham, MA) software. Quantification of VEC staining thickness was performed with the Integrated Morphometry Analysis module in MetaMorph software.
Images were analysed using MetaMorph Imaging System and Volocity (PerkinElmer, Waltham, MA) software. Quantification of VEC staining thickness was performed with the Integrated Morphometry Analysis module in MetaMorph software.