Dieter Brandner; Ginger Withers (2010) CIL:8783, 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.

本彩色合成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（使用罗丹明磷脂素）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结构稳定的轴心。详细方法：如前所述，制备胚胎大鼠海马神经元（参见Kaech和Banker，2006，Nat Protoc）。细胞如前所述进行荧光染色（Withers和Banker，1998，在《培养神经细胞》一书中，MIT Press）。简而言之，细胞经固定（4%甲醛，4%蔗糖磷酸盐缓冲液，pH 7.4，37°C，15分钟）、透化（0.25%透化剂Triton，7分钟）后，用针对微管蛋白的单克隆DM1A抗体（Sigma，Alexa 488偶联的二抗，Molecular Probes，激发光波长494，发射光波长519）和罗丹明标记的磷脂素（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、40倍镜头（HCX PL Fluotar，数值孔径0.75）、Leica GFP滤光片组（激发光波长BP 470/40；二色镜，500，抑制滤光片，BP 525/50）、Leica N3滤光片组（激发光波长BP546/12；二色镜，565，抑制滤光片，BP 600/40）的Leica DMRA显微镜和Photometrics CoolSnap ES CCD相机以及MetaMorph软件采集。合并图像使用MetaMorph的颜色组合功能生成。