Cultured hippocampal neurons after 17 days in vitro, immunostained for MAP2, a microtubule associated protein localized to dendrites (red) but not axons, which are not apparent in the immunofluorescence channel. Both axons, and dendrites, can be seen in the hidden phase micrograph which can be turned on using the edit function in the detailed viewer. Neurons at 10, 14, and 17 days in vitro are represented in this image group. Detailed methods: 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 (HM2, from Sigma with d549 conjugated secondary, excitation, 555, emission, 568, Jackson Immunoresearch). Images were acquired with a Leica DMRA microscope with a 40X lens (HCX PL Fluotar, NA 0.75), Photometrics CoolSnap ES CCD camera and MetaMorph software. A multilayer stack of the fluorescent image of MAP2 staining and the phase image was generated using MetaMorph.
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