Selective labeling of phosphatidylserine for cryo-TEM by a two-step immunogold method

Immunogold labeling in transmission electron microscopy (TEM) utilizes the high electron density of gold nanoparticles conjugated to proteins to identify specific antigens in biological samples. In this work we applied the concept of immunogold labeling for the labeling of negatively charged phospholipids, namely phosphatidylserine, by a simple protocol, performed entirely in the liquid-phase, from which cryo-TEM specimens can be directly prepared. Labeling included a two-step process using biotinylated annexin-V and gold-conjugated streptavidin. We initially applied it on liposomal systems, demonstrating its specificity and selectivity, differentiating between 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) membranes. We also observed specific labeling on extracellular vesicle samples isolated from THP1 cells and from MDA-468 cells, which underwent stimulations. Finally, we compared the levels of annexin-V labeling on the cells vs. on their isolated EVs by flow cytometry and found a good correlation with the cryo-TEM results. This simple, yet effective labeling technique makes it possible to differentiate between negatively charged and non-negatively charged membranes, thus shillucidating their possible EV shedding mechanism.