2.6. Immunocytochemistry

On day two after seeding, microvessels were washed with PBS for 5 minutes, fixed with 3.7% paraformaldehyde (Sigma) for 15 minutes, permeabilized with 0.1% Triton X-100 (Sigma) for 15 minutes, and blocked with 1% donkey serum (Sigma) overnight at 4°C. Microvessels were incubated for 6 hours at 4°C with primary antibodies (see Supplementary Table 1 for details) and for 20 minutes at room temperature with Alexa Flour-647 and Alexa Flour-488 secondary antibodies (Life Technologies). To localize nuclei or f-actin, 1:500 DAPI solution (Thermo Scientific) and 1:50 Alexa Fluor-647 phalloidin (Invitrogen) were added, respectively. Confocal z-stacks were obtained on a swept field confocal microscope system (Prairie Technologies) with illumination provided by MLC 400 monolithic laser combiner (Keysight Technologies). To fully reconstruct microvessels, approximately four hundred 0.4 μm slices were acquired using a 40x objective (Nikon).