Sc1836

The embedded brain tissues were sectioned to a thickness of 5 µm using a microtome (Leica, Nussloch, Germany). The sections were mounted on coated slides (#631-1349, Adhesion slides, Menzel Gläser, Polysine^®, Thermo Fisher Scientific, UK), hydrated, and boiled in citrate buffer (pH 6.0) for antigen retrieval. After washing twice with 0.1% Triton X-100/PBS for 5 min, the sections were blocked with 1.5% normal horse serum for 1 h at RT to inhibit non-specific signals. The sections were subsequently incubated overnight at 4°C with anti-VEGFA (1:200, #SC1836, Santa Cruz Biotechnology), anti-CD31 (1:200, # AF3628, R&D Systems, MN, USA), anti-p-MAPK1 (#9102, Cell Signaling Technology, Inc), and anti-SP-1 antibodies, followed by incubation with the appropriate secondary antibodies for 1 h at RT. The slides were observed using an Olympus BX53 fluorescence microscope with DP73 digital camera (Olympus, Tokyo, Japan). Three fields were arbitrarily photographed for each tumor, and the average value of the three fields was used as a representative value for one tumor. The fluorescence intensity was analyzed by Image J using the following formula: Corrected Total Cell Fluorescence (CTCF) = Integrated Density − (Area of selected cell × Mean fluorescence of background readings).

Total proteins were extracted from intestine using Pro-Prep™ lysis buffer (Intron Biotechnology, Seoul, Korea) and quantitated with Bradford assay reagent (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol. Immunoblots were probed using the following antibodies: VEGF (1:1000, sc-1836, Santa Cruz Biotechnology, CA, USA), eNOS (1:1000, sc-634, Santa Cruz Biotechnology) and β-actin (1:3000, Sigma, MO, USA). The blots were incubated with these antibodies overnight at 4 °C and detected by the luminescence method using ECL solution (NEL104001EA, PerkinElmer, Waltham, MA, USA).