CIL:10111, Rattus, multipolar neuron. In Cell Image Library

This multi-layer image shows the spatial relationship between filamentous actin (red) and microtubule array (green) in cultured hippocampal neurons, grown for 3 days 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. Some nonneuronal cells may also appear in the field.
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). Fluorescent and phase images were acquired with a Leica DMRA microscope with a mercury arc lamp, a 20X lens (HC PL Fluotar, NA 0.5), 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.

本多层图像展示了体外培养3天的海马神经元内，丝状肌动蛋白（filamentous actin，红色）与微管阵列（microtubule array，绿色）的空间分布关系。采用罗丹明鬼笔环肽（rhodamine phalloidin）进行的肌动蛋白染色，可标记沿轴突与树突分布的生长锥尖端及丝状伪足延伸结构；而微管染色则可显示这些神经突起的稳定骨干部分。视野中也可能出现部分非神经元细胞。

详细实验方法：
胚胎大鼠海马神经元的制备方法参照已有文献（参见Kaech与Banker，2006年，Nat Protoc）。神经元的荧光染色制备流程参照已有方法（Withers与Banker，1998年，收录于《神经细胞培养》（Culturing Nerve Cells），麻省理工学院出版社（MIT Press）出版）。
简言之，实验步骤如下：将细胞固定于含4%甲醛、4%蔗糖的磷酸盐缓冲液（pH 7.4）中，37℃孵育15分钟；随后用0.25% Triton透化处理7分钟；继而对微管蛋白进行免疫染色：使用单克隆抗体DM1A（Sigma公司），搭配Alexa 488标记的二抗（Molecular Probes公司，激发波长494 nm，发射波长519 nm）；同时使用罗丹明标记的鬼笔环肽（Molecular Probes公司，激发波长540 nm，发射波长565 nm）进行肌动蛋白染色。
荧光及相差图像通过以下设备采集：Leica DMRA显微镜，搭配汞弧灯、20倍物镜（HC PL Fluotar，数值孔径NA 0.5）；Leica GFP滤光片组（激发光BP 470/40；二向色镜500 nm，抑制滤光片BP 525/50）；Leica N3滤光片组（激发光BP546/12；二向色镜565 nm，抑制滤光片BP 600/40）；Photometrics CoolSnap ES CCD相机，以及MetaMorph成像软件。