Bs 1310r

The integrin antibody-blocking assay was done according to how it was described previously [19 (link)], with some modifications. Polyvinyl chloride microtiter plates (Corning, NY, USA) were coated with HM-3/HSA (400 μg/mL) overnight at 4 °C. The coated plates were washed two times with 0.9% sodium chloride and then blocked with serum-free RPMI medium containing 5% BSA for 1 h at 37 °C. After washing again, B16F10 cells (1 × 10⁴ cells) in 100 μL of serum-free 1640 medium were added to all wells and incubated for 2 h at 37 °C. Afterwards, the unbound cells were removed by washing the wells two times with serum-free 1640 medium. Adherent cells were fixed with 4% (w/v) paraformaldehyde for 1 h at RT, rinsed twice in PBS, and stained with 1% (w/v) crystal violet for 30 min at RT. Following two times of washing, the stained cells were counted. For the adhesion inhibiting assay, cell suspensions were incubated with the anti-α5β1 monoclonal antibody (Bioss bs-1310R; 1:500) or anti-αvβ3 monoclonal antibody (Sino Biological CT014-MM01, Beijing, China; 1:500) for 1 h at RT. Then, the assay was performed in the same way as the adhesion assay.

The tumors were fixed with Mildform 10 N (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) overnight. After cryoprotection in 30% sucrose, the frozen tissue slices of the tumors were sectioned (10 µm) using a cryostat and stained for immunohistochemical evaluation using an integrin α_vβ₃ polyclonal primary antibody (rabbit immunoglobulin G) (bs-1310R, Bioss Antibodies, Woburn, MA, USA) and an anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (Envision + system-HRP labeled polymer; K4003, Dako, Glostrup, Denmark). Following immunohistochemical labeling, the tissue slices were examined under a light microscope (BZ-9000, Keyence, Osaka, Japan).