Alexa fluor 546 donkey anti mouse antibody

Platelet adhesion and activation was assessed using immunofluorescence staining for the platelet marker, CD41 (Abcam, Cambridge, MA), and blood clot marker, tissue plasminogen activator (t-PA, Abcam, Cambridge, MA). Platelets were fixed in 4% paraformaldehyde (Fisher Scientific, Waltham, MA) for 15 minutes followed by incubation in 1% bovine serum albumin for 30 minutes to block nonspecific binding. Platelets were double-labeled as follows: substrates were first incubated with primary antibodies (t-PA), diluted 1:50 in PBS for 60 minutes followed by Alexa Fluor 546 donkey anti-mouse antibody (Invitrogen, Carlsbad, CA) diluted 1:100 in PBS for 60 minutes. Finally the samples were incubated with anti-human CD41 fluorescein isothiocyanate labeled mouse monoclonal antibody diluted 1:300 in PBS for 60 min. Four images were acquired per replicate using a Nikon Eclipse Ti-E motorized inverted microscope (Nikon Instruments INC., Melville, NY) to obtain a total of 12 images per substrate. The fluorescent intensity of the images was quantified using ImageJ^{18 , 19 , 20 (link)}.