Immunofluorescent analysis of connexin-43 using monoclonal antibodies to its extracellular domain

Immunofluorescent analysis of connexin-43 was carried out on preparations of fixed and living cultures of rat and human glioma cells, HEK293 cells, and frozen sections of the rat brain with experimental glioma using monoclonal antibodies to recombinant extracellular fragment of connexin-43 (E2 second extracellular loop). These monoclonal antibodies visualized membrane and cytoplasmic pools of connexin-43 in preparations fixed with paraformaldehyde. Incubation of monoclonal antibodies to E2 extracellular loop with living cells led to visualization of only connexin hemichannels on cell membranes. No immunofluorescence characteristic of dimer connexons, organizing the gap junction, was detected. This fact indicates that antibodies to connexin-43 extracellular loop E2, obtained in our study, specifically react with target antigen solely at the stage of connexon presentation on the membrane in the form of hemichannels. These monoclonal antibodies can be used for immunophenotyping and sorting of connexin-43-positive cells in vitro and as the guide molecules in addressed delivery of diagnostic preparations and drugs to glioma cells in vivo.