Deconvolution immunofluorescence microscopy analysis of platelets isolated from WT and FOG1 ki/ki mice. (A) Paraformaldhyde-fixed and permeabilized WT and FOG1 ki/ki platelets were labeled with antibodies to P-selection (P-sel; green) and to the lysosomal marker LAMP1 (red). For WT platelets, 3 separate studies are shown on left. Because of the weak P-selectin expression in FOG1 ki/ki platelets relative to WT cells when the images were taken at the same exposure and gain settings as in WT (top row for images 1 and 2 on left), the same P-selectin image was taken at a higher setting to provide a more comparable signal to WT (bottom row for images 1 and 2 on left). There was no obvious variation in LAMP1 levels. Longer exposure of the P-selectin label in FOG1 ki/ki platelets (bottom rows) reveals vesicular labeling that does not overlap with that for LAMP1. (B-C) Resting (top 2 rows) or thrombin-activated (10 minutes; bottom row) platelets were labeled for (B) P-selectin (green) and VWF (red) or (C) P-selectin (green) and PBP (red). P-selectin exposure was again increased for the FOG1 ki/ki and labeling partially overlaps labeling for both VWF and PBP in resting platelets from both WT and FOG1 ki/ki platelets. After thrombin treatment, P-selectin is mobilized to the cell surface in WT but not FOG1 ki/ki platelets. Note that levels of VWF and PBP expression are not substantially different in resting WT and FOG1 ki/ki platelets and do not substantially change on thrombin stimulation of FOG1 ki/ki platelets.