Alexaflour 488 secondary antibodies

Immunofluorescence staining of HMEC monolayers was performed using chamber slides (Fisher, Hanover Park, IL). Cells were cultured to monolayer, treated with either vector or 20 ng/ml VEGF or 50 ng/ml Ang-1 and co-treated with either 10 μM Triciribine (TCBN) or 1 μM pp-2 for 24 hours and washed twice with ice-cold PBS, fixed using 2% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 for 15 min, and blocked with 2% BSA in sterile PBS for 1 hour. Cell monolayers were then incubated with antibodies against VE-cadherin (1:100, Rabbit antibodies, Cell Signaling, Danvers, MA) at 4°C overnight. Immunofluorescence was revealed using goat anti-rabbit AlexaFlour-488 secondary antibodies (1:2000, Life Technologies, Grand Island, NY). Cells were mounted on to a glass slide using DAPI containing mounting medium (Vector Laboratories, PA). Images were captured using a confocal microscope equipped with argon and helium/neon lasers (LSM510, Zeiss, Germany). Controls were performed by omitting primary antibodies. All controls gave negative results with no detectable non-specific labeling.

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).