Allantois Dissection and Culture for Vascular Analysis

Allantoises were dissected from E8.5 embryos and cultured for 18 hours in 8-well µ-slides (Ibidi) coated with bovine fibronectin (Sigma) in DMEM (Gibco) supplemented with 10% fetal calf serum (Invitrogen) in a humidified tissue culture incubator at 37°C and 5% CO₂ (Sanyo). Inhibitors were made up as per manufacturer's instructions and added into the growth medium when setting up the culture. Control explants were vehicle treated. Inhibitors were obtained from Calbiochem and used at the following concentrations: LY294002, 10 µM; Y-27632, 10 µM. Allantois explants were fixed with 4% paraformaldehyde, followed by wholemount staining with rat anti-VE-cadherin antibody (BD Biosciences) together with rat anti-endomucin. Overlapping pictures of the explants were taken with an inverted epifluorescence microscope (Olympus Cell^R), making sure that no area was over-or underexposed. Photographs were assembled using the panorama function of Photoshop software.