Ginger Withers (2011) CIL:12442, 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天的培养，其胞体和树突分支上的突触前接触丰富。神经元经MAP2（微管相关蛋白，定位在树突上，绿色）和突触素I（突触前囊泡蛋白，红色）进行免疫染色。除免疫染色的突触前终端外，在此制备中轴突不可见。采用相对低倍率物镜（20倍）捕捉单个细胞整个树突分支。详细方法：参照Kaech和Banker（2006年，《自然实验方法》）所述方法制备胚胎大鼠海马神经元。细胞如前所述进行荧光染色（Withers和Banker，1998年，《培养神经细胞》，麻省理工学院出版社）。简而言之，细胞经4%甲醛、4%蔗糖磷酸盐缓冲液（pH 7.4）固定，用0.25%的Triton进行渗透处理，并分别对MAP2（单克隆HM2，Sigma，与Alexa 488偶联的二级抗体，激发光波长494，发射光波长519[Invitrogen，Molecular Probes]）和突触素I（来自P. DeCamilli，与DyLight549偶联的二级抗体，激发光波长555，发射光波长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软件获取。每个荧光体的单个图像经过着色并使用MetaMorph软件组装成堆栈文件。