Sc 8711

Tissue samples were obtained from patients with GBC who underwent resection at the Department of Surgery and Oncology, Kyushu University Hospitals, Fukuoka, Japan between 2001 and 2012. Approval for the use of tissues was obtained from patients in accordance with the Ethical Committees for Clinical Study at Kyushu University. Immunohistochemical staining was performed using 4-μm-thick formalin-fixed, paraffin-embedded tissue sections and primary antibodies for TrkB (1:50, sc-8316, Santa Cruz Biotechnology), MIB-1 (1:100, Dako), VEGF (1:100, sc-152, Santa Cruz Biotechnology), or HIF-1α (1:100, sc-8711, Santa Cruz Biotechnology). Endogenous peroxidase activity was blocked for 30 minutes using methanol containing 0.3% hydrogen peroxidase. Sections were incubated with primary antibodies overnight at 4°C, followed by incubation for 40 minutes with the secondary antibodies at room temperature. The reaction products were visualized using diaminobenzidine (DAB). The TrkB staining intensity of each slide was separately scored for the tumor invasion front and the tumor center as follows: 1 researcher and 1 pathologist evaluated the whole section on a blind basis and scored the staining intensity as none (0), weak (1), moderate (2), or strong (3) (Figure 1B 1a–1d).

Conditioned media from cell lines was collected by maintaining the cells in serum-free medium. Whole cell extract from tumor tissues was obtained by suspending the tumor tissue in lysis buffer composed of 20 mM Tris-HCl pH 8.0, 137 mM NaCl, 10% (w/v) glycerol, 1% Triton X-100, 2 mM Na₂VO₄, 2 mM EDTA and homogenizing using a homogenizer. The cell suspension was incubated on ice for 40 minutes with intermittent vortexing and centrifuged to collect the whole cell extract. Samples were resolved using SDS-PAGE, transferred to nitrocellulose membrane (Bio-Rad) and probed with rabbit anti-VEGFA (ABS82, Millipore; kindly provided by Balaji Krishnamachary, Johns Hopkins University School of Medicine, Baltimore, MD), rabbit anti-Lamin B (ab16048, Abcam), goat anti-HIF-1α (sc8711, Santa Cruz Biotechnology), and mouse anti-GAPDH (sc-47724, Santa Cruz; kindly provided by Dr. Shanmugasundaram Ganapathy Kanniappan, Johns Hopkins University, School of Medicine, Baltimore, MD) antibodies. Membranes were incubated with horseradish peroxidase-conjugated antibody against rabbit (NA934V, GE HealthCare), mouse (NA931V, GE HealthCare) or goat (NB7357, Novus Biologicals; kindly provided by Balaji Krishnamachary) IgG and binding was revealed by chemiluminescence detection (Millipore).

Slides with xenograft tumors and GBC cells from patients were immunostained with primary antibodies for TrkB (1:100, sc-8316, Santa Cruz Biotechnology) and HIF-1α (1:100, sc-8711, Santa Cruz Biotechnology), followed by Alexa Fluor 488-labeled and Alexa Fluor 594-labeled secondary antibodies (1:1000, Invitrogen), as described previously [28 (link)].