Dieter Brandner; Ginger Withers (2010) CIL:8778, 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（使用罗丹明荧光素）突显了轴突和树突上的生长尖端及伪足延伸，而微管染色则揭示了这些结构的稳定轴心。详细方法：按照前人所述（参见Kaech和Banker，2006，Nat Protoc）制备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《神经细胞培养》，MIT出版社）。简要来说，细胞被固定（4%甲醛，4%蔗糖磷酸盐缓冲液，pH 7.4，37°C，15分钟），透化（0.25%吐温，7分钟），并免疫染色微管蛋白（单克隆DM1A，Sigma，与Alexa 488偶联的二抗，Molecular Probes，激发光波长494，发射光波长519）以及罗丹明偶联的鬼笔毒环肽（Molecular Probes，激发光波长540，发射光波长565）。图像使用带有汞灯、40X镜头（HCX PL Fluotar，NA 0.75）、Leica GFP滤光片组（激发光通带BP 470/40；双色镜，500，抑制滤光片，BP 525/50）；Leica N3滤光片组（激发光通带BP546/12；双色镜，565，抑制滤光片，BP 600/40），Photometrics CoolSnap ES CCD相机和MetaMorph软件的合并图像功能获取。合并图像通过MetaMorph颜色合并功能生成。