Paraformaldehyde (pfa)

ECFCs were plated on rat tail collagen (50 μg/mL) coated glass coverslips. Three different staining procedures were used to detect intra‐ and/or extracellular VWF. Solely intracellular VWF was visualized after fixation and permeabilization with ice‐cold methanol, after which the cells were washed twice with PBS and blocked with PBS, 1% fetal bovine serum (Gibco) and 1% bovine serum albumin (Sigma‐Aldrich). Intra‐ as well as extracellular VWF was visualized after fixation with 4% paraformaldehyde (Alfa Aesar, Karlsruhe, Germany), after which the cells were washed once and blocked and permeabilized with PBS, 5% normal goat serum (DAKO, Glostrup, Denmark) and 0.02% saponin (Sigma‐Aldrich). Extracellular VWF was visualized after fixation with 4% paraformaldehyde, after which the cells were washed once and blocked with PBS and 5% normal goat serum (DAKO). Cells were stained for VWF with polyclonal antibody rabbit anti‐hVWF (A0082, DAKO) and for VE‐cadherin with purified mouse anti‐human CD144 (BD Biosciences) diluted in the corresponding blocking buffer. Nuclear staining was performed with Hoechst (Thermo Fisher Scientific) diluted in PBS. Coverslips were mounted by ProLong^® Diamond Antifade Mountant (Thermo Fisher Scientific) and cells were visualized by the Leica TCS SP8 X WLL converted confocal microscope equipped with a HC PL APO CS2 63×/1.40 OIL immersion objective.