Metamorph software 7

The cells were fixed with 4% paraformaldehyde, and were incubated with a goat polyclonal anti-FGFR-2 antibody (1:100) or a rabbit anti-VEGF-A antibody (1:200) at 4°C overnight. The cells were washed with PBS, then incubated with an Alexa 488-labeled anti-goat IgG or anti-rabbit IgG antibody (1:1000) with or without Alexa 568-labeled phalloidin (1:50) for 60 min. Fluorescent images were observed under a Digital Eclipse C1 TE2000-E confocal microscope (Nikon Insteck, Kanagawa, Japan), and analyzed using control software EZ-C1 (Nikon Insteck). Confocal settings, including the laser power and detector sensitivity, remained unchanged during the acquisition of all images. The images of 3-D culture were collected at 0.5-μm intervals with a laser to form a stack in the Z-axis, and were used to generate a 3-D image, using Volocity (Improvision, Coventry, UK). Total intensity of VEGF-A was measured by MetaMorph software 7.6 (Universal Imaging, Marlow, UK).