Platelet Activation Marker Detection

The CaP-blasted Ti and plain SUS316L plates were washed with PBS and the fixed platelets were microperforated with T-PBS (0.1% Tween-20-containing PBS) for 1 min [16 (link)]. The samples were washed twice with PBS and blocked with 0.1% Block Ace (Sumitomo Dainippon Pharma Co. Ltd., Osaka, Japan) in T-PBS for 1 h. The samples were then treated with mouse monoclonal anti-CD62P, anti-CD63 (1:100 dilution; BioLegend, San Diego, CA, USA), or anti-PDGF-B (1:200 dilution; Santa Cruz Biotechnology, Dallas, TX, USA) antibodies overnight at 4 °C. Post-treatment, the samples were again washed twice with T-PBS and subsequently probed with a secondary antibody (goat anti-mouse IgG H&L conjugated with Alexa Fluor® 488; Abcam, Cambridge, MA, USA) for 60 min along with phalloidin (Cytopainter Phalloidin-iFlour™ 555 Reagent; Abcam) at ambient temperature in the dark. Isotype controls for mouse primary antibodies (Abcam) were used as negative controls.
Finally, after washing with PBS, the samples were mounted using an antifade mounting medium (Vectashield®; Vector Laboratories, Burlingame, CA, USA), and target proteins were examined under a fluorescence microscope (Eclipse 80i; Nikon, Tokyo, Japan) connected to a cooled CCD camera (VB-7000; Keyence, Osaka, Japan) [22 (link)].