Primary mouse monoclonal anti vash1 antibody

Western blotting was performed as described previously. 31 Cells were lysed in cell lysis buffer containing 25 mM Tris (pH7.4), 100 mM NaCl, and 1% Tween 20. Equal amounts of protein were loaded onto 10% gels and separated by SDS-PAGE. Resolved proteins were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (BIO-RAD, Japan). The membranes were blocked with 5% low-fat dry milk in TBS-T [25 mM Tris (pH7.4), 125 mM NaCl, 0.4% Tween 20] for 1 h at room temperature, followed by incubation with 1:1000 dilutions of primary mouse monoclonal anti-EZH2 antibody (Dako, Tokyo, Japan) or primary mouse monoclonal anti-VASH1 antibody (Abcam Tokyo, Japan) at 4 C overnight. Blots were extensively washed with TBS-T and incubated with HRP-conjugated secondary antibody (1:2000 dilution; Santa Cruz Biotechnology, CA, USA) diluted in TBS-T for 1 h at room temperature. Membranes were washed and visualized using the chemiluminescent detection reagent kit (ECL Plus; GE Healthcare Corp., Tokyo, Japan).

Immunohistochemical staining was performed on 3-mm formalin-fixed, paraffin-embedded sections as described previously. 9, 11, 29, 30 In brief, endogenous peroxidase activity was blocked using 3% hydrogen peroxide for 5 min. The sections were incubated with primary mouse monoclonal anti-EZH2 antibody (1:50 dilution; Dako, Tokyo, Japan) or primary mouse monoclonal anti-VASH1 antibody (1:200 dilution; Abcam, Tokyo, Japan) overnight at 4 C, followed by incubation with a biotin-free horseradish peroxidase-labeled polymer of the Envision Plus detection system (Dako, Tokyo, Japan). A positive reaction was visualized with diaminobenzidine solution, followed by counterstaining with Mayer's hematoxylin. EZH2 and VASH1 staining was scored by counting the percentage of positively stained cells in five high-magnification fields. The cutoff value defined as the median percentage of stained cells (10% for EZH2, 35% for VASH1) was used to classify low expression and high expression.