Hc pl apo cs2 63x 1

Cells were plated on glass coverslips coated with collagen I in 24 well plates and grown to 3 to 5 days postconfluency. In Leiden, cells were fixed with 4% paraformaldehyde (PFA; Alfa Aesar) without washing, then rinsed with PBS and blocked and permeabilized for 20 minutes in blocking buffer (PBS; 5% Normal Goat Serum [Dako]; 0.02% saponin [Sigma‐Aldrich]). In Kingston, cells were washed once with PBS and fixed with Fixation Solution (BD Bioscience). Cells were rinsed with PBS and permeabilized with PBS/0.1% Triton x‐100 for 10 minutes on ice, then washed with PBS and blocked for 20 minutes with Protein Block (Dako).
After fixation, permeabilization, and blocking, cells were stained with primary antibodies for VWF and VE‐cadherin (Table S1 in supporting information) diluted in either blocking buffer or PBS/1% bovine serum albumin (BSA) (Sigma‐Aldrich). Nuclear staining was performed with either Hoechst (Thermo Fisher Scientific) or DAPI (Sigma‐Aldrich) diluted in PBS. Coverslips were mounted by Mounting Media (Dako) or ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) and cells were imaged using the Leica TCS SP8 inverted confocal microscope (Leica Microsystems) with the white light laser (WLL), hybrid detectors (HyD), and the HC PL APO CS2 63x/1.40 oil immersion objective. Images were acquired and analyzed using the LAS‐X Software (Leica Microsystems).