Dieter Brandner; Ginger Withers (2010) CIL:8773, 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在体外培养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。通过罗丹明磷光素标记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的稳定杆部。详细方法：如前所述（参见Kaech和Banker，2006，Nat Protoc），制备了胚胎大鼠海马神经元。细胞如前所述进行荧光染色（Withers和Banker，1998，在《培养神经细胞》，MIT Press）。简而言之，细胞用4%甲醛、4%蔗糖的磷酸盐缓冲盐水（pH 7.4，37°C，15分钟）固定，用0.25%的Triton（7分钟）渗透，并用DM1A单克隆抗体（Sigma）标记的Alexa 488偶联的二级抗体以及罗丹明标记的磷光素（Molecular Probes，激发光波长494，发射光波长519）进行免疫染色。图像使用配备汞灯弧光灯、40X镜头（HCX PL Fluotar，数值孔径0.75）、Leica GFP滤光片组（激发光波段BP 470/40；二色镜500，抑制滤光片BP 525/50）、Leica N3滤光片组（激发光波段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。合并图像使用MetaMorph的色彩组合功能生成。