CIL:10114, 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 5 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5天的海马神经元中，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）进行肌动蛋白染色，可标记轴突与树突上的生长尖端及丝状伪足突起；而微管染色则可显示这些神经突起的稳定干段。视野中亦可能存在少量非神经元细胞。
详细实验方法：
胚胎大鼠海马神经元的制备参照既往报道流程（参见Kaech与Banker，2006年，《自然实验方案》（Nat Protoc））。荧光染色样本的制备同样参照既往方法（Withers与Banker，1998年，载于《神经细胞培养》（Culturing Nerve Cells），麻省理工学院出版社（MIT Press））。
简言之，实验人员对细胞进行固定处理：将细胞置于含4%甲醛、4%蔗糖的磷酸缓冲盐溶液（pH 7.4）中，37℃孵育15分钟；随后进行透化处理：使用0.25% Triton（曲拉通）孵育7分钟；继而开展双重标记染色：针对微管蛋白使用单克隆抗体DM1A（Sigma公司），搭配Alexa 488标记的二抗（Molecular Probes公司，激发波长494nm，发射波长519nm）；同时使用罗丹明标记的鬼笔环肽（Molecular Probes公司，激发波长540nm，发射波长565nm）标记肌动蛋白。
荧光图像与相差图像通过徕卡DMRA显微镜采集，该设备配置汞弧灯、20倍物镜（HC PL Fluotar，数值孔径NA 0.5）、徕卡GFP滤光片组（激发光BP 470/40；二向色镜500；阻断滤光片BP 525/50）与徕卡N3滤光片组（激发光BP546/12；二向色镜565；阻断滤光片BP 600/40），并搭配Photometrics CoolSnap ES电荷耦合器件（CCD）相机及MetaMorph软件完成图像采集。