Immunogold Labeling of Extracellular Vesicles

A drop (5μl) of _SEVs (isolated by UC) suspended in PBS was deposited on formvar-carbon-coated electron microscopy grids for 20 min at room temperature, fixed with 2% paraformaldehyde in 0.2 M phosphate buffer (pH 7.4), for 20 min at room temperature, and post fixed with 1% glutaraldehyde in PBS for 5 min at room temperature. Grids containing sEV were then washed and then blocked for 5 min at room temperature in blocking buffer (PBS, 1% BSA). sEVs were then immunolabelled with a mouse anti-human CD63 primary antibody (Abcam ab23792) diluted in blocking solution for 1 hour at room temperature, washed with PBS, 0,1% BSA, incubated with a rabbit antibody against mouse Fc fragment (Dako Agilent Z0412) in PBS 0,1% BSA for 20 min at room temperature. The preparations were then immunogold labeled with protein-A gold-conjugates (10 nm; Cell Microscopy Center, Department of Cell Biology, Utrecht University). Grids were analyzed on a Tecnai Spirit G2 electron microscope (Thermo Fischer Scientific) and digital acquisitions were made with a 4k CCD camera (Quemesa, Soft Imaging System). Images were analyzed with iTEM software (iTEM CE Olympus serie) and data with Prism-GraphPad Prims software (v8)⁶⁷ (link).