CIL:8728, Rattus, multipolar neuron. In Cell Image Library

Rat hippocampal neurons grown in vitro, fixed after 23 days in culture, and immunostained for MAP2 (red) to reveal the dendritic arbor, and localization of α Calcium Calmodulin Kinase II (green), a multifunctional kinase that has high concentrations at excitatory postsynaptic sites and presynaptic terminals, and is involved in regulating synaptic plasticity.
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 alpha-CamKII (clone 6G9, Abcam, with Alexa 488 conjugated secondary, excitation, 494, emission, 519 [Invitrogen, Molecular Probes]) and MAP2 (Ab266 from S. Halpain, 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. This merged image was generated with the MetaMorph color combine function.

本数据集包含体外培养的大鼠海马神经元：于培养23天后完成固定，通过免疫荧光染色标记微管相关蛋白2（MAP2，红色）以显示树突分支，同时标记α-钙调蛋白依赖性激酶II（α Calcium Calmodulin Kinase II）——该多功能激酶在兴奋性突触后位点与突触前末梢中高丰度富集，参与调控突触可塑性。
详细实验方法如下：
胚胎大鼠海马神经元的制备参照已发表方案（参见Kaech与Banker，2006年，《自然实验方案》（Nat Protoc））。细胞的荧光染色样本制备流程同样参照既往发表方法（Withers与Banker，1998年，收录于《神经细胞培养》（Culturing Nerve Cells），麻省理工学院出版社）。
简言之，实验步骤为：将细胞置于预热至37℃的4%甲醛、4%蔗糖磷酸缓冲盐溶液（pH 7.4）中固定15分钟；随后用0.25% Triton透化处理7分钟；接着进行双重免疫荧光染色：针对α-CamKII，使用Abcam公司的6G9克隆抗体，辅以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品牌）；针对微管相关蛋白2（MAP2），使用S. Halpain馈赠的Ab266抗体，辅以DyLight549标记的二抗（激发波长555 nm，发射波长568 nm，购自Jackson Immunoresearch）。
图像采集采用Leica DMRA正置荧光显微镜，搭配汞弧灯光源、40倍物镜（HCX PL Fluotar，数值孔径NA 0.75）、Leica GFP滤光片组（激发光带通BP 470/40；二向色镜500；抑制滤光片带通BP 525/50）与Leica N3滤光片组（激发光带通BP546/12；二向色镜565；抑制滤光片带通BP 600/40），辅以Photometrics CoolSnap ES型CCD相机及MetaMorph图像分析软件完成。本合并图像通过MetaMorph软件的色彩合并功能生成。