Dieter Brandner; Ginger Withers (2010) CIL:8785, 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培养的神经元中纤维状肌动蛋白（红色）与微管阵列（绿色）的空间关系，该神经元在体外培养1天后形成。使用罗丹明-鬼笔毒环素进行的肌动蛋白染色突出了轴突和树突上的生长尖端和丝状突起，而微管染色则揭示了这些过程稳定的中轴。详细方法：如前所述（参见Kaech和Banker，2006年，《Nat Protoc》），制备了胚胎大鼠海马神经元。细胞按照前述方法进行荧光染色（参见Withers和Banker，1998年，《培养神经细胞》，MIT Press）。简而言之，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），并免疫染色管蛋白（单克隆DM1A，Sigma，与Alexa 488偶联的二抗，Molecular Probes，激发光，494，发射光，519）和罗丹明偶联的鬼笔毒环素（Molecular Probes，激发光，540，发射光，565）。图像使用带有汞弧灯、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显微镜采集。合并图像通过MetaMorph色彩组合功能生成。