Resolving sub-synaptic compartments with double immunofluorescence labeling in hippocampal neurons

Immunofluorescence microscopy of synaptic proteins is a powerful and commonly used approach in cellular neurosciences. Many studies use green/red color overlays of immunofluorescence images to demonstrate synaptic co-localization of an unknown protein with a known synaptic marker. However, this approach fails to identify the specific sub-synaptic compartment in which a protein is localized. Here we describe a novel analysis method to determine the precise location of proteins within synapses of cultured hippocampal neurons with double immunofluorescence staining. This approach is based on center-to-center distance measurements of fluorescent clusters of protein pairs coexisting in synapses. We validated the method by analyzing the distances between different combinations of well-established synaptic marker proteins. The results demonstrate that protein pairs in the active zone and the postsynaptic density, two sub-synaptic compartments which are separated by less than 50 nm, can be readily distinguished from each other and from marker pairs co-localized within a single sub-synaptic compartment. Thus, center-to-center distance analysis can resolve the distance across the synaptic cleft and it is useful for localizing synaptic proteins to specific pre- and postsynaptic compartments.