Dieter Brandner, Ginger Withers (2011) CIL:8467, Rattus, multipolar neuron. CIL. Dataset

Development of axon and dendritic arbors in cultured hippocampal neurons after 2 days in vitro. At this timepoint, many, but not all cells are polarized, evidenced by the absence of a long, slender axon. MAP2 staining (red) is becoming 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，神经元轴突和树突的发育情况经过2天后。在此时间点，许多细胞已实现极化，但并非所有细胞均呈现出长而细的轴突。MAP2染色（红色）正逐渐集中于树突，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（例如胶质细胞）较为罕见，但亦可能存在于该制备物中。
详细方法：按照先前描述（参见Kaech和Banker，2006，Nat Protoc）制备胚胎大鼠海马神经元。细胞按照先前描述进行荧光染色（参见Withers和Banker，1998，在《培养神经细胞》，MIT Press）。简而言之，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进行通透化，并通过免疫染色对微管蛋白（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）进行染色。图像通过Leica DMRA显微镜、40X（HCX PL APO，NA 1.25）镜头、Photometrics CoolSnap ES CCD相机和MetaMorph软件获取。此合并图像是通过MetaMorph颜色组合功能生成的。