Synapse development in cultured hippocampal neurons after 15 days in vitro. Neurons were immunostained for MAP2, a microtubule associated protein localized to dendrites (green) and the presynaptic vesicle protein Synapsin I (red) to show the density of presynaptic contacts along the dendritic arbor.
Embryonic rat hippocampal neurons were prepared as previously described (see Kaech and Banker, 2006, Nat Protoc). Cells were prepared for fluorescent staining as previously described (Withers and Banker, 1998, in Culturing Nerve Cells, MIT Press). Briefly, cells were fixed (4% formaldehyde, 4% sucrose in phosphate buffered saline, pH 7.4), permeabilized with 0.25% Triton and immunostained for MAP2 (monoclonal HM2, Sigma, with Alexa 488 conjugated secondary, excitation, 494, emission, 519 [Invitrogen, Molecular Probes]) and synapsin I (from P. DeCamilli, with DyLight549 conjugated secondary, excitation, 555, emission, 568, [Jackson Immunoresearch]). Images were acquired with a Leica DMRA microscope with a 20X objective (Fluotar, 0.5NA), Photometrics CoolSnap ES CCD camera and MetaMorph software. This color was generated using the MetaMorph "color combine" function.
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