Dieter Brandner; Ginger Withers (2010) CIL:10260, Rattus, multipolar neuron. CIL. Dataset

Early stages of dendritic development and synapse formation in cultured hippocampal neurons. This multilayer image shows neurons fixed at 5 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.

该数据集展示了培养的海马神经元树突发育及突触形成的早期阶段。该多层图像呈现了在体外培养5天时固定的神经元，经免疫染色定位到树突上的蛋白MAP2（绿色）和突触囊泡蛋白Synapsin I（红色）。视野中的未染色轴突在相位通道中清晰可见，该通道虽为隐藏状态，但在观察者中可开启。图像组中展示了体外培养3天、5天和7天的神经元。