Immunogold Labeling of Phosphatidylserine by Annexin V in Cryo-TEM Specimens

Maayan Nir-Shapira, The Norman Seiden Multidisciplinary Program for Nanoscience and Nanotechnology, The Department of Chemical Engineering, and the Russell Berrie Nanotechnology Institute (RBNI), Technion, Haifa, Israel
Naama Koifman, The Norman Seiden Multidisciplinary Program For Nanoscience And Nanotechnology, The Department Of Chemical Engineering, And The Russell Berrie Nanotechnology Institute (rbni), Technion, Haifa, Israel
Yeshayahu Talmon, The Department Of Chemical Engineering, Technion, Haifa, Israel

In the last few decades the use of colloidal gold in transmission electron microscopy (TEM) has grown at an enormous rate. Immunogold labeling takes advantage of the high electron density of gold conjugated to antibodies, which in turn adsorb to a specific antigen. Although the probing molecules are often antibodies, other proteins of a specific affinity can be tagged.

Phosphatidylserine (PS) is a negatively charged phospholipid, found mostly in the inner leaflet of the cell membrane, facing the cytoplasm. Certain cell processes, such as apoptosis and microparticle shedding, involve PS migration to the outer leaflet. This phenomenon can be studied using the cellular protein, annexin V, which has high affinity to PS. Annexin V binding is Ca²⁺ dependent, which should be taken into account during the procedure.

Liposomes are spheroidal vesicles, composed of one or more lipid bilayers. They allow the easy and fairly realistic mimicking of bio-membranes. Immunogold labeling of PS in mixed lipid bilayer will significantly contribute to the study of the nano-scale domain formation mechanism. Until now these domains have been studied by direct imaging only on the micrometer scale, not on the nanoscopic scale. Understanding this phenomenon is important, because they are involved in a variety of cellular functions and biological events.

Our work presents immunogold labeling in cryo-TEM. Cryo-TEM preserves the liposomes as close as possible to their native state, thus providing a more reliable view of the nanostructure and its morphology. We attempted to label PS liposomes prepared by sonication and extrusion. Labeling was performed in solution, using biotinilated annexin V and gold-conjugated streptavidin. We optimized the working conditions leading to extensive labeling of PS. The optimized labeling protocol has been applied to extracellular vesicle samples, emphasizing the simplicity and ease of use of this developed methodology.