Dieter Brandner;Ginger Withers (2010) CIL:2891, Rattus, multipolar neuron. CIL. Dataset

Development of the axon and dendritic arbors in cultured hippocampal neurons after 2 days in vitro. MAP2 staining (red) highlights the dendrites, while microtubule staining (green) reveals both the axons and dendrites in the field of view. Neurons at 2, 3, 5 and 7 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 tubulin (monoclonal DM1A, from Sigma with Alexa 488 conjugated secondary, excitation, 494, emission, 519 [Invitrogen, Molecular Probes]) and MAP2 (polyclonal Ab266, from S. Halpain with d549 conjugated secondary, excitation, 555, emission, 568, Jackson Immunoresearch). Images were acquired with a Leica DMRA microscope with a 20X (Fluotar, NA 0.5) lens, Photometrics CoolSnap ES CCD camera and MetaMorph software. Image generated with the MetaMorph color combine function.

在体外培养的海马神经元中，轴突和树突的发育情况于2天后得以呈现。MAP2染色（红色）突显了树突，而微管染色（绿色）则揭示了视野中的轴突与树突。该图像组展示了体外培养2、3、5及7天时的神经元。详细方法：如前所述（参见Kaech和Banker，2006年，《Nat Protoc》），制备了胚胎大鼠海马神经元。细胞按照前述方法进行荧光染色（参见Withers和Banker，1998年，《Culturing Nerve Cells》，MIT Press）。简言之，细胞经4%甲醛、4%蔗糖磷酸盐缓冲液（pH 7.4）固定，用0.25%的Triton进行透化，并免疫染色管蛋白（单克隆DM1A，Sigma公司产品，Alexa 488偶联二抗，激发波长494，发射波长519 [Invitrogen，Molecular Probes]）和MAP2（多克隆Ab266，S. Halpain公司产品，d549偶联二抗，激发波长555，发射波长568，Jackson Immunoresearch）。图像使用Leica DMRA显微镜配备20X（Fluotar，NA 0.5）镜头、Photometrics CoolSnap ES CCD相机和MetaMorph软件采集。图像通过MetaMorph色彩组合功能生成。