Dieter Brandner; Ginger Withers (2010) CIL:10355, Rattus, multipolar neuron. CIL. Dataset

Cultured hippocampal neurons after 14 days in vitro, immunostained for MAP2, a microtubule associated protein localized to dendrites (red) but not axons, which are not apparent in the immunofluorescence channel. Both axons, and dendrites, can be seen in the hidden phase micrograph which can be turned on using the edit function in the detailed viewer. Neurons at 10, 14, and 17 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), permeabilized with 0.25% Triton and immunostained for MAP2 (HM2, from Sigma with d549 conjugated secondary, excitation, 555, emission, 568, Jackson Immunoresearch). Images were acquired with a Leica DMRA microscope with a 40X lens (HCX PL Fluotar, NA 0.75), Photometrics CoolSnap ES CCD camera and MetaMorph software. A multilayer stack of the fluorescent image of MAP2 staining and the phase image was generated using MetaMorph.

培养于体外14天的海马神经元，经MAP2（微管相关蛋白，主要定位于树突，呈红色）免疫染色，轴突未染色，在免疫荧光通道中不可见。轴突和树突在隐藏相显微照片中清晰可见，可通过详细查看器中的编辑功能打开。图中展示了体外培养10天、14天和17天的神经元。详细方法：如前所述制备胚胎大鼠海马神经元（参见Kaech和Banker，2006年，Nat Protoc）。细胞按照前述方法进行荧光染色（参见Withers和Banker，1998年，《培养神经细胞》，MIT Press）。简言之，细胞以4%甲醛、4%蔗糖磷酸盐缓冲液（pH 7.4）固定，用0.25%的Triton处理通透化，并经MAP2（HM2，Sigma产品，d549偶联二抗，激发波长555，发射波长568，Jackson Immunoresearch）进行免疫染色。使用Leica DMRA显微镜（40X镜头，HCX PL Fluotar，NA 0.75）、Photometrics CoolSnap ES CCD相机和MetaMorph软件获取图像。使用MetaMorph生成MAP2染色荧光图像和相位图像的多层堆栈。