Dieter Brandner, Ginger Withers (2011) CIL:8455, 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，神经轴突和树突分支的发育经过3天后。在此时间点，大多数细胞已实现极化，这可由长而细的轴突的缺失得到证实。MAP2染色（红色）正逐渐在树突中分离，而微管染色（绿色）则揭示了视野内的轴突和树突。非神经元细胞（例如胶质细胞）较为罕见，但在此制备中也可能存在。详细方法：按照先前描述的方法制备胚胎大鼠海马神经元（参见Kaech和Banker，2006年，《Nat Protoc》）。细胞按照先前描述的方法进行荧光染色（Withers和Banker，1998年，《Culturing Nerve Cells》，MIT Press）。简而言之，细胞被固定（4%甲醛，4%蔗糖在磷酸盐缓冲盐溶液中，pH 7.4），用0.25%的Triton进行通透化，并免疫染色管蛋白（单克隆DM1A，来自Sigma，与Alexa 488偶联的二级抗体，激发波长494，发射波长519 [Invitrogen，Molecular Probes]）和MAP2（多克隆Ab266，来自S. Halpain，与d549偶联的二级抗体，激发波长555，发射波长568，Jackson Immunoresearch）。图像使用Leica DMRA显微镜，40X（HCX PL APO，NA 1.25）镜头，Photometrics CoolSnap ES CCD相机和MetaMorph软件采集。此合并图像由MetaMorph颜色组合功能生成。