Mapping GPIIb/IIIa on murine blood platelets with expansion microscopy

Mapping GPIIb/IIIa on murine blood platelets with expansion microscopy

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 (https://doi.org/10.1101/2021.02.16.431449)

Here we provide a selection of typical images of receptor distributions on unexpanded and expanded murine platelets, the code for simulation of platelet receptor distributions including a user manual and a video protocol of 10x expansion microscopy with resting platelets.

Sample images: 

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 -> high colocalization

Case II: directly immunolabeled with a monoclonal antibody targeting GPIIb/IIIa (MWReg30) carrying either Alexa Fluor 594 or Alexa Fluor 488) -> 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) -> low colocalization

The provided sample images are .ims (Imaris) files contain the raw data, chromatic aberration corrected data & 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.

MatLab Scipts: 3D-Image simulation of Platelet Receptor Distributions

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.

Video Protocol: 10x expansion microscopy with resting platelets.

The provided video protocol describes the sample preperation for 10x expansion microscopy of resting platelets, including platelet washing, immunolabeling, gelation, digestion, expansion and sample mounting for imaging. Detailed step by step protocols are also provided in the supplementary material (https://doi.org/10.1101/2021.02.16.431449).