Confocal immunodetection of TF and GPIbα in human, nonpermeabilized platelets. After fixation, blockade of nonspecific binding sites, and overnight incubation at 4°C, EDTA-PGE1 platelets were immunolabeled with polyclonal anti-TF and/or α-GPIbα (α-CD42b, clone SZ2; Immunotech, Westbrook, ME). For TF, incubation with rabbit anti-sheep IgG was required before incubating with the appropriate FITC antibody. Alexa 555 (Invitrogen) was used to visualize the GPIbα. Top row shows, from left to right: nonstimulated, freshly prepared platelets labeled for TF, GPIbα, and merge imaging. Bottom row shows an aliquot of the same sample stimulated with TRAP and collagen for 2 hours. A striking enhancement of both labels is noticed with a significant increase in the colocalization of both proteins. No leukocytes were observed in all the experiments.
