Mapping GPIIb/IIIa on murine blood platelets with expansion microscopy

<strong>Mapping GPIIb/IIIa on murine blood platelets with expansion microscopy</strong> We visualize the membrane architecture of platelets by mapping platelet integrin GPIIb/IIIa by super-resolution fluorescence. Our approach combines colocalization and expansion microscopy and successfully interrogates highly abundant receptors and their interactions where conventional methods fail in such dense 3D scenarios. Here we provide a s<em>election of typical images</em> of receptor distributions on unexpanded and expanded murine platelets and the <em>code</em> for simulation of platelet receptor distributions including a <em>user manual.</em> <strong>Sample images: </strong> Unexpanded, 4x expanded and 10x expanded resting murine platelets on glycine coated coverslips were imaged on a SP5 confocal microscope (detailed methods can be found in the manuscript). There are three different labeling combinations: Case I: directly immunolabeled with monoclonal antibodies antibodies carrying Alexa Fluor 488 (MWReg30, green channel) or Alexa Fluor 594 (JON6, magenta channel) can bind simultaneously on the same GPIIb/IIIa receptor -&gt; high colocalization Case II: directly immunolabeled with a monoclonal antibody targeting GPIIb/IIIa (MWReg30) carrying either Alexa Fluor 594 or Alexa Fluor 488) -&gt; low colocalizaiton Case III: directly immunolabeld with monoclonal antibodies carrying Alexa Fluor 488 targeting GPIIb/IIIa (JON6) and carrying Alexa Fluor 594 targeting GPIX (p0p6) -&gt; low colocalization The provided sample images are .ims (Imaris) files contain the raw data, chromatic aberration corrected data &amp; deconvolved data as consecutive time points, as well as the cropped image stack of a single platelet with the masked data used for colocalization analysis. <strong>MatLab Scipts: 3D-Image simulation of Platelet Receptor Distributions</strong> The provided MatLab scripts allow to create 2-color, 3D image stacks based on simulated platelet receptor distributions and the image resolution provided by confocal microscopy and sample expansion. Refer to the user manual for a detailed description of how to set up and run the simulation.