CIL:10253, Rattus, multipolar neuron. In Cell Image Library

Early stages of dendritic development and synapse formation in cultured hippocampal neurons. This multilayer image shows neurons fixed at 3 days in vitro and immunostained for the dendritically localized protein MAP2 (green) and the presynaptic vesicle protein Synapsin I (red). Unstained axons in the field are evident in the phase channel, which is hidden but can be turned on in the viewer. Neurons at 3, 5 and 7 days in vitro are represented in this image group.

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, warmed to 37°C prior to fixation, 15 minutes), permeabilized with 0.25% Triton (7 minutes) and immunostained for MAP2 (monoclonal HM2, Sigma, with Alexa 488 conjugated secondary, excitation, 494, emission, 519 [Invitrogen, Molecular Probes]) and synapsin I (from P. DeCamilli, with DyLight549 conjugated secondary, excitation, 555, emission, 568, [Jackson Immunoresearch]). 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.

培养海马神经元中树突发育与突触形成的早期阶段。该多层成像图展示了体外培养（in vitro）第3天的固定神经元，其中树突定位蛋白MAP2（绿色）与突触前囊泡蛋白Synapsin I（红色）已完成免疫染色标记。视野内未染色的轴突可在相差通道中清晰显现，该通道默认隐藏，可在查看器中开启。该图像组涵盖了体外培养3、5、7天的神经元样本。

详细实验方法：胚胎大鼠海马神经元的制备参照已发表方案（详见Kaech与Banker，2006年，《Nature Protocols》）。细胞的荧光染色制备流程参照已发表方法（Withers与Banker，1998年，收录于《培养神经细胞》，麻省理工学院出版社）。简要而言，细胞固定流程为：采用预热至37℃的含4%甲醛、4%蔗糖的磷酸盐缓冲液（pH 7.4）固定15分钟；随后用0.25% Triton透化处理7分钟；随后针对MAP2与Synapsin I进行免疫染色：MAP2使用单克隆抗体HM2（Sigma公司），搭配Alexa 488标记的二抗（激发波长494 nm，发射波长519 nm，购自Invitrogen旗下Molecular Probes）；Synapsin I抗体由P. DeCamilli实验室提供，搭配DyLight549标记的二抗（激发波长555 nm，发射波长568 nm，购自Jackson Immunoresearch）。图像采集使用Leica DMRA显微镜，配备汞弧灯、40倍物镜（HCX PL Fluotar，数值孔径0.75）、Leica GFP滤光片组（激发光BP 470/40；二向色镜500；抑制滤光片BP 525/50）与Leica N3滤光片组（激发光BP546/12；二向色镜565；抑制滤光片BP 600/40），搭配Photometrics CoolSnap ES CCD相机及MetaMorph软件完成采集。