Dieter Brandner; Ginger Withers (2010) CIL:8786, Rattus, multipolar neuron. CIL. Dataset

This color combined image shows the spatial relationship between filamentous actin (red) and microtubule array (green) in cultured hippocampal neurons, grown for 1 day in vitro. Actin staining (with rhodamine phalloidin) highlights the growing tips and filopodial extensions along axons and dendrites, while microtubule staining reveals the stable shafts of these processes.
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, 37°C, 15 minutes), permeabilized (0.25% Triton, 7 minutes) and immunostained for tubulin (monoclonal DM1A, Sigma, with Alexa 488 conjugated secondary, Molecular Probes, excitation, 494, emission, 519) and rhodamine-conjugated phalloidin (Molecular Probes, excitation, 540, emission, 565). Images were acquired with a Leica DMRA microscope with a mercury arc lamp, a 40X lens (HCX PL Fluotar, NA 0.75), Leica GFP filter set (excitation, BP 470/40; dichromatic mirror, 500, suppression filter, BP 525/50); Leica N3 filter set (excitation, BP546/12; dichromatic mirror, 565, suppression filter, BP 600/40), Photometrics CoolSnap ES CCD camera and MetaMorph software. Merged image was generated with the MetaMorph color combine function.

本彩色组合图像展示了体外培养1天的海马神经元中，丝状肌动蛋白（红色）与微管阵列（绿色）之间的空间关系。肌动蛋白染色（采用罗丹明-鬼笔毒环素）突出了轴突和树突上的生长尖端及伪足扩展，而微管染色揭示了这些结构稳定轴干。详细方法：按照先前描述的方法（参见Kaech和Banker，2006年，《Nat Protoc》），制备了胚胎大鼠海马神经元。细胞按照先前描述的方法进行荧光染色（参见Withers和Banker，1998年，《培养神经细胞》，MIT出版社）。简要来说，细胞用（4%甲醛，4%蔗糖在磷酸盐缓冲盐溶液中，pH 7.4，37°C，15分钟）固定，用（0.25% Triton，7分钟）渗透化，并使用DM1A单克隆抗体（Sigma，与Alexa 488偶联的二级抗体，Molecular Probes，激发光波长494，发射光波长519）和罗丹明偶联的鬼笔毒环素（Molecular Probes，激发光波长540，发射光波长565）进行免疫染色。图像使用配备汞灯弧光灯、40X镜头（HCX PL Fluotar，NA 0.75）、Leica GFP滤光片组（激发光，BP 470/40；二色镜，500，抑制滤光片，BP 525/50）；Leica N3滤光片组（激发光，BP546/12；二色镜，565，抑制滤光片，BP 600/40）、Photometrics CoolSnap ES CCD相机和MetaMorph软件的Leica DMRA显微镜获取。合并图像通过MetaMorph色彩组合功能生成。