Fluorescein isothiocyanate fitc conjugated donkey anti goat igg

PRP was obtained from WT or β3^−/− mice and were pre-incubated with anti-β3 integrin mAb M1 (where indicated) for flow cytometry. Resting or activated (20 µM or 50 µM ADP or 5 µg/mL collagen) platelets were fixed with 4% paraformaldehyde at 4 °C. Samples were incubated with goat anti-human apoA-IV antibody (N-20, Santa Cruz), which also detects mouse apoA-IV, and then incubated with fluorescein isothiocyanate (FITC)-conjugated donkey anti-goat IgG (Santa Cruz) at room temperature in the dark. Phosphate-buffered saline (PBS, 0.2 mL) was added to the sample immediately before acquisition. All samples were analyzed by flow cytometry using a calibrated FACScan™ flow cytometer (BD Biosciences). For Western blot analysis, resting or activated (0.5 U/mL thrombin) gel-filtered mouse platelets (4 × 10⁷ to 1 × 10⁸) were lysed, separated by SDS-polyacrylamide gel electrophoresis (PAGE), transferred onto polyvinylidene fluoride (PVDF) membrane, and probed with polyclonal goat anti-human apoA-IV antibody and rabbit anti-goat IgG HRP conjugated antibody (Bio-Techne). Following apoA-IV detection, actin was probed as a loading control after stripping the blots and the actin bands were visualized using HRP conjugated goat anti-mouse actin antibody (Supplementary Fig. 16).