Af467

Cells were fixed in 4% paraformaldehyde/PBS for 10 minutes at room temperature and rinsed with PBS 1X. For intracellular staining, cells were permeabilized with 0.1% Triton X100/PBS for 10 minutes at room temperature. Cells were incubated over night at +4°C with antibodies anti-CD144 (1∶200, Beckman Coulter, IM1597), anti-CD31 (1∶20, BD Pharmingen, 550389), anti-ZO1 (1∶200, BD Biosciences, 610966), anti-CL3 (1∶100, Abcam, C0144), anti-CL5 (1∶100, Invitrogen, 34–1600), anti-OCCL (1∶100, Invitrogen, 33–1500), anti-EFNB2 (1∶10, R&D Systems, AF467), anti-NRP1(1∶20, R&D Systems, AF3870), anti-HEY2 (1∶50, Santa Cruz Biotechnology, sc-28747), anti-ANGPT2 (1∶20, R&D Systems, AF623), anti-CXCR4 (1∶100, Abcam, ab2074), anti-COUP-TFII (1∶100, R&D systems, PP-H7147-10), anti-EPHB4 (1∶10, R&D systems, AF3038) or anti-NRP2 (1∶40, R&D Systems, AF2215) antibodies diluted in 3% BSA/PBS and, after 5 PBS 1X washes, labeled with secondary antibodies Alexa Fluor 488 IgG (Invitrogen, Cergy-Pontoise, France). Cells were then stained with 2 µg/mL of 40,6-diamidino-2-phenylindole (DAPI) and examined with a DMR fluorescence microscope (Leica, Rueil Malmaison, France) equipped with a CoolSnap HQ2 camera (Photometrics, Tucson, AZ) controlled by MetaVue® Analyzing Software (Molecular Devices LLC, Sunnyvale, CA).

Sections or coverslips with cells were washed with Tris buffered saline (TBS) and incubated in blocking buffer (1% glycine, 0.4% Triton X-100, 3% BSA, 0.1% sodium azide and 10% normal goat serum in TBS) for one hour at room temperature. Sections or coverslips were incubated in the primary antibodies in species-appropriate combinations for 24 hours at 4 °C. Primary antibodies and dilution used in our experiments included rabbit anti-GFP (Catalog: A-11122, 1:1500, Invitrogen), rat anti-GFP (Catalog: GF090R, 1:2000, Nacalai), mouse anti-MAP2 (Catalog: ab11267, 1:200, Abcam), Goat anti-EphB2 (Catalog: AF467, 1:250, R&D systems), rabbit anti-Ephrin-A5 (Catalog: ab70114, 1:250, abcam) and rabbit anti-RFP (Catalog: ab62341, 1:250, Abcam). After incubation with primary antibodies, sections or coverslips were washed with TBS and incubated with secondary antibodies in blocking buffer at room temperature for two hours. Secondary antibodies included Alexa 488, 546, 633 conjugated goat anti-mouse, anti-rat, anti-rabbit IgG (1:500; Abcam). Sections or coverslips were then washed with TBS and mounted with ProLong Gold anti-fade (Invitrogen). All images were taken by Zeiss LSM700 confocal microscopy. The volume, migratory distance, and the average fluorescent intensity were estimated by using ImageJ (U. S. National Institutes of Health).