Fixed platelets on the cp-Ti plates were directly subjected to treatment with primary antibodies, such as mouse monoclonal anti-CD62P antibody (1:20 dilution; BioLegend, San Diego, CA, USA), rabbit polyclonal anti-PDGF-B (1:200 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-TGFβ1 (1:200 dilution; Santa Cruz Biotechnology), rabbit monoclonal anti-PPARγ (1:100 dilution; Cell Signaling Technology, Danvers, MA, USA), and mouse monoclonal anti-fibrin antibodies (1:400 dilution; GeneTex, Inc., Irvine, CA, USA) overnight at 4 °C. In this study, based on our preliminary observations that our routine blocking or washing with Tween-20-containing PBS also visualized intra-platelet binding of the antibodies we used, we did not perform blocking or use such a detergent-containing PBS in the main experiments in this study.
The samples were then probed with the corresponding secondary antibodies or non-immunized rabbit IgG (Life Technologies Corporation, Carlsbad, CA, USA) as an isotype control for 60 min at ambient temperature in the dark. The samples were finally mounted using an antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA, USA) and examined under a fluorescence microscope (ECLIPSE 80i; Nikon) connected with a cooled CCD camera (VB-7000; Keyence) [23 (link)].
The samples were then probed with the corresponding secondary antibodies or non-immunized rabbit IgG (Life Technologies Corporation, Carlsbad, CA, USA) as an isotype control for 60 min at ambient temperature in the dark. The samples were finally mounted using an antifade mounting medium (Vectashield; Vector Laboratories, Burlingame, CA, USA) and examined under a fluorescence microscope (ECLIPSE 80i; Nikon) connected with a cooled CCD camera (VB-7000; Keyence) [23 (link)].
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