Sc 8316

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).

Western blotting was performed as described previously [28 (link)]. The protein-transferred membranes were incubated overnight at 4°C with primary antibodies for TrkB (1:200, sc-8316, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-TrkB (1:500, ab197072, Cambridge, MA, USA), BDNF (1:200, sc-546, Santa Cruz Biotechnology), MMP-2 (1:200, sc-10736, Santa Cruz Biotechnology), MMP-9 (1:200, sc-6840, Santa Cruz Biotechnology), E-cadherin (1:200, sc-7870, Santa Cruz Biotechnology), vimentin (1:200, sc-6260, Santa Cruz Biotechnology), SLUG (1:200, sc-15391, Santa Cruz Biotechnology), SNAIL (1:200, sc-10433, Santa Cruz Biotechnology), twist (1:200, sc-15393, Santa Cruz Biotechnology), VEGF-C (1:200, sc-7133, Santa Cruz Biotechnology), VEGF-D (1:200, sc-7603, Santa Cruz Biotechnology), p-MEK-1/2 (1:200, sc-7995, Santa Cruz Biotechnology), Erk1/2 (1:200, No9102 Cell signaling Technology), p-Akt1/2/3 (1:200, sc-101629, Santa Cruz Biotechnology), or Akt1/2/3 (1:200, sc-8312, Santa Cruz Biotechnology). Peroxidase-linked secondary antibodies (Amersham Biosciences, Piscataway, NJ, USA) were subsequently added and the membranes were further incubated for 1 h at room temperature. The antibodies for α-tubulin (1:1000, Sigma-Aldich, St. Louis, MO, USA) and β-actin (1:200, sc-47778, Santa Cruz Biotechnology) were used as protein loading controls.

The paraffin sections of laryngeal cancer were deparaffinized and rehydrated routinely. The sections were immersed with 3% H₂O₂ and 10% goat serum and they were subsequently incubated overnight with a primary antibody for TrkB (sc-8316, Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:100) at 4°C. The samples were incubated with HISTOFINE simple stain MAX-PO (R) (Nichirei, Tokyo, Japan) and finally visualized by 3,3’diaminobenzidine (DAB) with hematoxylin-counterstain.

Lysates were separated by SDS-PAGE (4%–12% Bis-Tris, Invitrogen, Carlsbad, CA, USA) and transferred to PVDF membrane and probed with antibodies against TrkB (1:1,000, sc-8316, SantaCruz Biotechnology, Santa Cruz, CA, USA) and pTrkB^s478 (1:1,000, R-1718-50, Biosensis), p44/42 MAPK (ERK1/2, 1:1,000, #9102 Cell Signaling Technologies, Danvers, MA, USA) and phosphorylated ERK1/2 (pERK1/2, 1:1,000, #9101 Cell Signaling Technologies, Danvers, MA, USA). All blots shown are representative of at least three independent experiments. Optical density value for each band was determined using FIJI/ImageJ and corrected to loading control and normalized against the relevant control condition.

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)].