Fitc conjugated sheep anti rabbit igg

A rabbit polyclonal antibody against zebrafish αIIb was custom ordered from Alpha Diagnostic Intl. Inc. (San Antonio, TX). 2 μl of blood collected, as described above, was placed into 100 μl of 4% paraformaldehyde. The cells were incubated for 1 hour and then washed with PBS three times by pelleting the cells in an Eppendorf centrifuge at 10,000 rpm and finally suspending them in 0.5 ml of PBS. To this cell suspension, 0.5 μg αIIb antibody was added and mixed by finger tapping. Nonimmune rabbit IgG was used as a negative control. After incubation for 1 hour at 20°C in primary antibodies, the cells were pelleted and washed twice with PBS. The pellet was suspended in a mixture of 100 μl of PBS containing 0.3 μg FITC-conjugated sheep anti-rabbit IgG (Sigma-Aldrich, St. Louis, MO). The cells were again washed in PBS, briefly rinsed with distilled water, and then suspended in 500 μl dH2O. These cells were analyzed using flow cytometry by either a Becton-Dickinson FACSCalibur or BD Accuri C6 Plus Flow Cytometer. For flow cytometry counting of unlabeled thrombocytes the blood collected in PBS described above is used directly. In all these experiments the several events were collected for one minute and the percentages of cells or microparticles were calculated after appropriate gating.