Dieter Brandner; Ginger Withers (2010) CIL:8776, 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.

此彩色组合图像展示了培养的海马神经元中细丝状肌动蛋白（红色）与微管阵列（绿色）的空间关系，神经元在体外培养1天后形成。肌动蛋白染色（使用罗丹明-鬼笔环肽）突出了轴突和树突上的生长尖端及伪足的延伸，而微管染色则揭示了这些过程的稳定杆状结构。
详细方法：按照先前描述的方法制备了胚胎大鼠海马神经元（参见Kaech和Banker，2006，Nat Protoc）。细胞按照先前描述的方式进行荧光染色（参见Withers和Banker，1998，在《培养神经细胞》一书中，MIT Press）。简要来说，细胞经过固定（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%吐温，7分钟），并对微管蛋白（单克隆DM1A，Sigma，与Alexa 488偶联的二级抗体，Molecular Probes，激发波长494，发射波长519）以及罗丹明偶联的鬼笔环肽（Molecular Probes，激发波长540，发射波长565）进行免疫染色。图像使用配有汞弧灯、40X镜头（HCX PL Fluotar，数值孔径0.75）、Leica GFP滤光片组（激发波长BP 470/40；双色镜，500，抑制滤光片，BP 525/50）；Leica N3滤光片组（激发波长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Leica DMRA显微镜采集。合并图像通过MetaMorph的颜色组合功能生成。