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.
Di Biase
Valentina
V
Department of Physiology and Medical Physics, Innsbruck Medical University, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria.
Flucher
Bernhard E
BE
Obermair
Gerald J
GJ
D000818:Animals; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006624:Hippocampus / Q000166:cytology*; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence / Q000379:methods; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D017729:Presynaptic Terminals / Q000737:chemistry / Q000378:metabolism; D013569:Synapses / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
D009419:Nerve Tissue Proteins