Dieter Brandner; Ginger Withers (2010) CIL:8782, 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）的空间关系。肌动蛋白染色（使用罗丹明标记的鬼笔环肽）突出了轴突和树突上的生长尖端和伪足扩展，而微管染色揭示了这些过程的稳定杆状结构。
详细方法：参照先前描述（参见Kaech和Banker，2006年，《自然方法》），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年，《培养神经细胞》，麻省理工学院出版社）。简要来说，细胞经固定（4%甲醛，4%蔗糖在磷酸盐缓冲盐溶液中，pH 7.4，37°C，15分钟），透化（0.25%透化剂，7分钟），并用抗微管蛋白抗体（单克隆DM1A，Sigma，与Alexa 488偶联的二级抗体，Molecular Probes，激发波长494，发射波长519）和罗丹明标记的鬼笔环肽（Molecular Probes，激发波长540，发射波长565）进行免疫染色。图像使用莱卡DMRA显微镜，配备汞弧灯、40倍镜头（HCX PL Fluotar，数值孔径0.75）、莱卡GFP滤光片组（激发波长BP 470/40；双色镜，500，抑制滤光片，BP 525/50）；莱卡N3滤光片组（激发波长BP546/12；双色镜，565，抑制滤光片，BP 600/40），Photometrics CoolSnap ES CCD相机和MetaMorph软件获取。合并图像使用MetaMorph颜色组合功能生成。