Dieter Brandner, Ginger Withers (2011) CIL:8277, Rattus, multipolar neuron. CIL. Dataset

Development of axon and dendritic arbors in cultured hippocampal neurons after 3 days in vitro. At this timepoint, most, but not all cells are polarized, evidenced by the absence of a long, slender axon. MAP2 staining (red) is becoming well segregated to the dendrites, while microtubule staining (green) reveals both the axons and dendrites in the field of view. Nonneuronal cells (e.g. glia) are infrequent, but may also be present in this preparation.

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 40X (HCX PL APO, NA 1.25) lens, Photometrics CoolSnap ES CCD camera and MetaMorph software. This merged image was generated with the MetaMorph color combine function.

在体外培养的海马神经元中，神经轴突和树突状枝条的发育情况于3天后得以观察。至此阶段，多数细胞已实现极化，此点可通过缺少细长的轴突得以证实。MAP2染色（红色）已良好地分离至树突中，而微管染色（绿色）则揭示了视野中的轴突和树突。非神经元细胞（如胶质细胞）虽较少见，但亦可能存在于本制备中。
详细方法：胚胎大鼠海马神经元制备过程参照前人描述（参见 Kaech 和 Banker，2006 年，Nat Protoc）。细胞荧光染色方法亦如前所述（参见 Withers 和 Banker，1998 年，《神经细胞培养》，MIT Press）。简要来说，细胞经固定（4% 甲醛，4% 蔗糖在磷酸盐缓冲盐水中，pH 7.4）、0.25% 吐温处理以实现通透化，并采用针对微管蛋白（单克隆DM1A，Sigma公司生产，Alexa 488偶联的二抗，激发波长494，发射波长519 [Invitrogen，Molecular Probes]）和MAP2（多克隆Ab266，S. Halpain公司生产，d549偶联的二抗，激发波长555，发射波长568，Jackson Immunoresearch）的免疫染色。图像采集使用Leica DMRA显微镜配备40X（HCX PL APO，NA 1.25）物镜，Photometrics CoolSnap ES CCD相机和MetaMorph软件。此合并图像由MetaMorph颜色组合功能生成。