Anti cd31 antibody

For immunoblot, antibodies to SEMA3F (ab39956) and NRP-1 (ab81321) were purchased from Abcam (Cambridge, UK). Antibodies to NRP-2 (#3366), PLEXIN-A1 (#3813), Myc-Tag (#2276), phospho-ERK (#4370), ERK (#9102), phospho-p70S6K (#9234), p70S6K (#9202) and cleaved caspase-3 (#9664) were from Cell Signaling Technology (Beverly, MA, USA) and the antibody to α-Tubulin (T6074) was from Sigma-Aldrich (St. Louis, Mo, USA).
For immunohistochemistry, the antibody for SEMA3F (HP035008) was purchased from Atlas Antibodies. Anti-phospho-p70S6K (sc-7984) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-chromogranin A (M0869), anti-human Ki67 (MIB1 clone, M7240) and anti-mouse Ki67 (TEC-3 clone, M7249) were from Dako (Glostrup, Denmark). Anti-CD31 antibody was from AnaSpec (#53332) (Fremont, CA, USA). For immunofluorescence, the antibody to phospho-Histone H3 (pH-H3) was from Cell Signaling Technology (#9706).

Immunohistochemistry was performed on an automated immunostainer (Ventana Discovery XT, Roche, Meylan, France) using DABmap Kit according to the manufacturer’s instructions. Staining was visualized with DAB solution with 3,3′-diaminobenzidine as a chromogenic substrate. The sections were counterstained with Gill’s hematoxylin. 4-μm tissue sections were preincubated with 1% goat serum then incubated with a rabbit polyclonal anti-CD31 antibody (AnaSpec, Fremont). To measure tumor-cell proliferation, tumor sections were incubated with a rabbit polyclonal anti–Ki-67 antibody (MIB-1, Dako, Trappes, France). Image analysis was performed by using a light microscope (Eclipse E400, Nikon France, Champigny, France) equipped with a tri-CDD video camera (Sony, Japan). Tumor microvessel density was quantified, as previously described4 (link)5 (link). The mitotic index was expressed as the percentage of Ki-67–positive nuclei. Ki67 analysis was performed using Histolab® software (Microvision Instruments, Evry, France).