Mouse anti human cd31

Whole cell lysates were prepared with 1% Nonidet P-40 (NP-40) lysis buffer (50 mM Tris-HCL, PH 7.4, 10% glycerol, 200 mM NaCl and 2 mM MgCl2) containing protease inhibitors. Protein samples were loaded onto 8%–15% SDS-PAGE. Membranes were blocked with 5% non-fat milk in 1X TBS wash buffer containing 0.3% Tween-20, then incubated with the following primary antibodies overnight at 4°C: rabbit anti-human VEGFR2, mouse anti-human CD31 (Santa Cruz Biotechnology, Santa Cruz, CA, United States); rabbit anti-human Bmi-1, PDGFR-α, phosphor-PDGFR-β, PDGFR-β, phosphor-Tie-2, Tie-2, phosphor-AKT, AKT (Cell Signaling, Danvers, MA, United States); mouse anti-human smooth muscle actin-α (SMA-α), mouse anti-GAPDH (Millipore/Sigma). Affinity-purified secondary antibodies conjugated with horseradish peroxidase (Jackson Laboratories, West Grove, PA, United States) were used and immunoreactive proteins were visualized by SuperSignal West Pico chemiluminescent substrate (Thermo Scientific, Rockford, IL, United States).

We collected pulp tissues from extracted non-carious human third molars. We also retrieved DPSC, SHED, and HDMEC from cell culture flasks at 80% confluency. Cells and pulp tissues were lysed in 1% NP-40 buffer. Then, the proteins were electrophoresed in SDS-polyacrylamide gel and transferred to nitrocellulose membranes. The membranes were incubated overnight at 4 °C with anti-human SEMA4D (Bioss Inc., Woburn, MA, USA), anti-human Plexin B1, or anti-human β-actin antibody conjugated with horseradish peroxidase (HRP) (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). To verify the endothelial differentiation, we incubated the membranes with the following primary antibodies: rabbit anti-human vascular endothelial growth factor receptor 1 or 2 (VEGFR1, VEGFR2), anti-human Tie2, or a mouse anti-human CD31 (Santa Cruz Biotechnology Inc.). The next day, membranes were incubated with affinity-purified secondary antibodies conjugated with HRP (Jackson Laboratories, West Grove, PA, USA). Lastly, the proteins were visualized using SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Rockford, IL, USA).

Fresh frozen sections were blocked with 3% BSA, and then, rabbit antiphospho-S6 ribosomal protein (Ser 235/236) antibody (Cell Signaling Technology), combined with mouse antihuman CD31 (Santa Cruz) or goat antihuman synaptopodin (Santa Cruz), rabbit antihuman CD3 (Abcam) combined with mouse antihuman CD4 and CD8 (Abcam) and interleukin (IL)-17A conjugated with PE (BD Biosciences) were added and incubated overnight at 4°C, followed by the secondary antibodies Alexa Fluor 488-labelled donkey antirabbit IgG, Alexa Fluor 647-labelled donkey antimouse IgG (Abcam) or TRITC-labelled donkey antigoat IgG (Invitrogen) for 30 min at 37°C. Nuclei were stained with 4′,6-diamidino-2-phenylindole (ZSGB-Bio). For negative controls, primary antibodies were replaced by PBS. Fluorescence images were acquired with fluorescence microscopy (DM2500; Leica, Germany).