Quantitative codistribution data indicate a stochastic pattern to the intermixing of α-granule proteins in resting human platelets. Resting human platelets were isolated and the distribution of 13 different platelet α-granule content proteins determined by pair-wise immunolabeling followed by Z-series spinning disk confocal microscopy. (A-L) Qualitative distribution of representative high, medium, and low codistribution pairings: (A-D) thrombospondin and platelet factor 4; (E-H) fibrinogen and thrombospondin; (I-L) fibrinogen and plasminogen. (A-L) Images are maximum intensity projections of confocal image stacks. Note that the XZ images are stretched in the Z dimension because of the approximately 3-fold lower resolution of confocal microscopy in the Z than XY dimension. (M,M′) 3D-SIM of human platelets stained for fibrinogen (Fibr, green channel) and VWF (red channel). (M′) Two times higher magnification. Arrows point to examples of zoned fluorescence within an apparently continuous structure (red vs yellow, green vs red). Single-plane XZ image slices are shown. (N-P) The quantitative confocal microscopy outcomes are tabulated in the right-hand column and then plotted in panel N. (N) Quantitative codistribution of the respective content protein pairings color-coded for proangiogenic (green) and antiangiogenic (red) properties. There is little, if any, codistribution trend based on physiologic function. (O) Quantitative codistribution of the 23 pairings plotted as a histogram displaying a near Gaussian pattern. (P) Quantitative codistribution data indicate that there is little, if any, variation in the extent of fibrinogen and endostatin colocalization between persons. Micrograph image details: Figure 1A-L,N-P; Microscope, Zeiss Axiovert 200M (Carl Zeiss); Objective lens, Zeiss planapochromat 100×/1.40NA oil objective, Imaging medium, buffered Mowiol containing 1% N-propylgallate as anti-fade reagent, Camera QImaging Retiga EXi (QImaging); Image acquisition software, iVision-Mac (Biovision Technologies) Version 4.0.16; Image deconvolution and colocalization software, Huygens Professional (Scientific Volume Imaging) Version 3.6. Figure 1M, M'; Microscope, OMXa (construction, design and software, University of California San Fransisco); Objective lens, Olympus UPlanSApo 100×/1.40 NA oil objective, Camera, Andor iXon3 897 EMCCD; Image acquisition and color channel alignment software,¹⁰  University of California San Fransisco, simplex algorithm for channel alignment. All pairings are quantified for a minimum of 30 individual platelets, averaged, and presented as the ± SEM.