Ginger Withers (2011) CIL:12556, Rattus, multipolar neuron. CIL. Dataset

Presynaptic contacts are abundant on both cell bodies and the dendritic arbor of cultured hippocampal neurons after 15 days in vitro. Neurons were immunostained for MAP2, a microtubule associated protein localized to dendrites (green) and synapsin I (red), a presynaptic vesicle protein. With the exception of the immunostained presynaptic terminals, axons are not visible in this preparation. A relatively low power objective (20X) was used to capture the entire dendritic arbor of individual cells.
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), permeabilized with 0.25% Triton 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 20X (HC Fluotar, NA 0.5) lens, Photometrics CoolSnap ES CCD camera and MetaMorph software. Individual images of each fluorophore were colorized and assembled as a a stack file using MetaMorph software.

在体外培养的海马神经元中，经过15天培养后，其细胞体和树突丛中存在丰富的突触前接触。神经元经MAP2（一种定位于树突的微管相关蛋白，呈绿色）和突触素I（一种突触前囊泡蛋白，呈红色）进行免疫染色。在本制备中，除免疫染色的突触前末端外，轴突不可见。采用相对低倍镜（20倍）以捕捉单个细胞的整个树突丛。
详细方法：参照先前描述的方法制备胚胎大鼠海马神经元（参见Kaech和Banker，2006年，《自然方法》）和荧光染色（参见Withers和Banker，1998年，《培养神经细胞》，麻省理工学院出版社）。简要来说，细胞用4%甲醛、4%蔗糖在磷酸盐缓冲盐溶液（pH 7.4）中固定，用0.25%的Triton进行通透化，并分别用MAP2（单克隆HM2，Sigma，Alexa 488偶联的二级抗体，激发波长494，发射波长519[Invitrogen，分子探针]）和突触素I（来自P. DeCamilli，DyLight549偶联的二级抗体，激发波长555，发射波长568[Jackson Immunoresearch]）进行免疫染色。图像使用Leica DMRA显微镜，20倍（HC Fluotar，NA 0.5）镜头，Photometrics CoolSnap ES CCD相机和MetaMorph软件采集。每个荧光标记的单个图像被着色并使用MetaMorph软件组装成一个堆栈文件。