Subcellular localization of PDI. Fixed nonstimulated and stimulated platelets were prepared for IEM. Immunogold labeling was carried out on frozen thin sections using 10-nm protein A gold conjugates, following standard procedures. Electron micrographs were obtained with a JEOL 1200EX electron microscope. For IF, platelets were allowed to settle on poly-L-lysine–coated coverslips, and PDI was detected after permeabilization with 0.5% Triton X-100. IF images were recorded on a Leitz DMIRB fluorescence microscope, interfaced with a Leica TCSNT confocal laser scanning microscope, using a 63×/1.4 Plan-Apo objective. (A-B) Nonstimulated platelets. Immunogold labeling with 2 different antibodies (panels A and B, respectively) gives the same results. indicate PDI localization within electron-dense tubular membranes. (Inset) Confocal image of endogenous PDI localization in permeabilized resting platelet. (C-D) Activation with 0.5 U/mL thrombin showing platelet shape change and loss of α-granules. No release of PDI to the OCS or translocation to the cell surface is observed. (Bottom left) Semiquantitative analysis of the endogenous PDI distribution. Secondary markers were rabbit anti–mouse Cy3 (IF) and 10-nm protein A gold (IEM). α indicates α-granules. Scale bars represent 500 nm (A,C) or 200 nm (B,D).