Receptor-mediated and fluid-phase cargoes took different endocytic routes in platelets. (A) Washed WT and VAMP-3−/− platelets (1.0 × 109/mL) were incubated ex vivo with Alexa 647-Fg and Oregon Green 488-Dextran at final concentrations of 1 µM each and incubated at 37°C for 1 to 30 minutes and prepared for 3D-structured illumination microscopy imaging as described in Methods. (B) WT and VAMP-3−/− mice were injected with Alexa 647-Fg and Oregon Green 488-Dextran at a concentration of 2 µM each per fluorophore through the retroorbital sinus. Twenty-four hours postinjection, platelets were harvested and prepared for 3D-structured illumination microscopy imaging. Slides from panels A and B were then imaged using the Nikon Ti-E N-STORM/N-SIM super-resolution microscope, and images were processed using the NIS-Elements v3.2 N-SIM/STORM suite software. Scale bars, 5 µm. Data are representative of at least 2 independent experiments. (C) Pearson’s correlation coefficients were calculated using the NIS-Elements v3.2 N-SIM/STORM suite software to show overlap between Alexa 647-Fg and Oregon Green 488-Dextran (depicted in white) at the indicated points. Graph shows the mean ± SEM of 2 independent experiments, with at least 30 cells per field, taken over 3 to 4 fields per time. Statistical analyses were performed using Student t test; n.s., not significant.
