Direct Immunofluorescence Staining Label 12 x 75-mm tubes. Add 20 L of CD61 PerCP to all tubes. Add 20 L of isotype control or platelet activation-dependent monoclonal antibody to the appropriately labeled test tubes. To demonstrate specific PAC-1 binding, include one tube with 10 L RGDS solution in the staining mixture. Within 10 minutes of blood collection, using a fresh micropipettor tip each time, carefully add 5 L of the unstimulated or activated whole blood suspension to the bottom of each tube.
Exercise care to prevent blood from running down the side of the tube.
If blood remains on the side of the tube, it may not be stained with the reagent. Discard and try again. Gently swirl the tubes to mix. Do not vortex. Incubate for 15 to 20 minutes at room temperature in the dark.