Dieter Brandner; Ginger Withers (2010) CIL:8775, 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培养的神经元中，丝状肌动蛋白（红色）与微管阵列（绿色）之间的空间关系，这些神经元在体外培养了1天。利用罗丹明标记的肌动蛋白进行的肌动蛋白染色突出了轴突和树突上的生长尖端和伪足延伸，而微管染色则揭示了这些过程的稳定杆状结构。
详细方法：参照前人方法（见Kaech和Banker，2006，Nat Protoc）制备胚胎大鼠海马神经元。细胞按照前人描述进行荧光染色（Withers和Banker，1998，在《培养神经细胞》一书中，MIT Press）。简要来说，细胞经固定（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%十二烷基硫酸钠，7分钟）后，进行针对微管蛋白的免疫染色（单克隆DM1A，Sigma，与Alexa 488偶联的二抗，Molecular Probes，激发波长494，发射波长519）以及罗丹明标记的鬼笔环肽（Molecular Probes，激发波长540，发射波长565）。图像使用配备汞灯弧光灯、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；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。