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 (polyclonal Ab266, S. Halpain, with DyLight549 conjugated secondary, excitation, 555, emission, 568, [Jackson Immunoresearch]) and tubulin (DM1A, Sigma with Alexa 488 conjugated secondary, excitation, 494, emission, 519 [Invitrogen, Molecular Probes]). Images were acquired with a Leica DMRA microscope with a 20X lens (Fluotar, NA 0.5), Photometrics CoolSnap ES CCD camera and MetaMorph software. This color was generated using the MetaMorph "color combine" function.