Dieter Brandner; Ginger Withers (2010) CIL:8779, 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.

本彩色合并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（使用罗丹明标记的鬼笔环肽）突显了轴突和树突上的生长尖端和伪足扩展，而微管染色则揭示了这些过程的稳定杆部。详细方法：如前所述制备胚胎大鼠海马神经元（参见Kaech和Banker，2006年，《Nat Protoc》）。细胞如前所述进行荧光染色（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%蔗糖磷酸盐缓冲液，pH 7.4，37°C，15分钟），透化（0.25%吐温，7分钟）后，进行针对微管蛋白的免疫染色（单克隆DM1A，Sigma，使用Alexa 488偶联的二抗，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）。图像使用配备汞弧灯、40X镜头（HCX PL Fluotar，NA 0.75）、Leica GFP滤光片组（激发波长BP 470/40；二色镜，500；抑制滤光片，BP 525/50）、Leica N3滤光片组（激发波长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显微镜获取。合并图像通过MetaMorph颜色合并功能生成。