Extracellular vesicles (EVs) are nano-sized, membrane-bound structures secreted by cells and play critical roles in mediating intercellular signaling. EVs based on their size as well as mechanisms of biosynthesis are categorized as either microvesicles (200–1000 nm) or exosomes (30–200 nm). The EVs carry several biomolecules like proteins, DNAs, RNAs, and lipids into other cells and modulate several cellular functions. Being of very small sizes, it is very challenging to analyze them using conventional microscopes. Here, we report a new method developed by us for visualizing EVs using simple immune-fluorescence based technique, wherein the isolated EVs can be stained with fluorescently tagged antibodies to proteins present in EVs. The stained EVs can then be analyzed by using either confocal or super-resolution microscopes. Our method detailed here is equally effective in staining proteins that are present inside the EVs as well as those localized to the membranes of vesicles. By employing unique staining strategies, we have minimized the background noise and thereby improved the signal strength in confocal microscope. Using electron microscopy, we have ascertained that the structural integrity of the labeled EVs is intact. More importantly, the labeling of EVs does not affect their functionality and their localization can be tracked after its uptake by recipient cells without resorting to any conventional reporter-based strategies or lipophilic dyes. In conclusion, the method described here is a simple, sensitive and efficient immune-fluorescence based method for visualization of molecules within the EVs.

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