Mouse anti human vwf

Cultures were fixed in 4% formaldehyde for 30 min, permeabilized with 0.5% TritonX-100 (Sigma) in PBS for 20 min, and blocked in 3% bovine serum albumin (BSA) in PBS for 1 hr. Detection of α-smooth muscle actin (αSMA), calponin, and von Willebrand Factor (vWF) in cultures was then performed in sequence. To identify SMCs, cultures were incubated with mouse anti-human αSMA (Invitrogen, 1:200 dilution) for 90 min with agitation, followed by incubation goat anti-mouse AlexaFluor® 488 (Invitrogen, 3 μg/mL). Calponin, a marker of SMC differentiation, was labeled using mouse anti-human calponin (Dako, 1:50 dilution) for 90 min with agitation, followed by incubation with goat anti-mouse AlexaFluor® 546 (Invitrogen, 3 μg/mL). Finally, vWF, an EC marker, was identified via mouse anti-human vWF (Dako, 1:25 dilution) for 90 min with agitation and goat anti-mouse AlexaFluor® 647 (Invitrogen, 3 μg/mL). Samples were washed for 20 min with agitation between each step to ensure removal of the previous antibody. Samples were visualized using a Nikon AR1 Multiphoton confocal microscope with 60x objective. Scans were completed with a xy area of 1028 μm² and one stack, 50 μm (1 μm per step) in the z-direction, was taken at ten separate locations in each culture.