Dieter Brandner; Ginger Withers (2010) CIL:10232, Rattus, multipolar neuron. CIL. Dataset

This multi-layer image shows the spatial relationship between filamentous actin (red) and microtubule array (green) in cultured hippocampal neurons, grown for 5 days in vitro. Actin staining (with rhodamine phalloidin) highlights the growing tips and filopodial extensions along axons and dendrites, while microtubule staining reveals the stable shafts of these processes. Some nonneuronal cells may also appear in the field. Neurons at 1, 3 and 5 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, 37°C, 15 minutes), permeabilized (0.25% Triton, 7 minutes) and immunostained for tubulin (monoclonal DM1A, Sigma, with Alexa 488 conjugated secondary, Molecular Probes, excitation, 494, emission, 519) and rhodamine-conjugated phalloidin (Molecular Probes, excitation, 540, emission, 565). Fluorescent and phase 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.

本多层图像展示了体外培养5天的海马神经元中，细丝状肌动蛋白（红色）与微管阵列（绿色）之间的空间关系。肌动蛋白染色（采用罗丹明偶联素）突出了轴突和树突上的生长尖端和伪足延伸，而微管染色揭示了这些过程的稳定杆部。视野中也可能出现一些非神经元细胞。本图像组展示了体外培养1天、3天和5天的神经元。