Dieter Brandner; Ginger Withers (2010) CIL:10227, 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 3 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.

本多层级图像展示了在体外培养3天的海马神经元中，丝状肌动蛋白（红色）与微管阵列（绿色）之间的空间关系。通过罗丹明标记的肌动蛋白染色，突触末梢和轴突、树突上的丝状突起得到突出显示，而微管染色则揭示了这些过程稳定的干结构。视野中可能也包含一些非神经元细胞。图中展示了体外培养1、3和5天的神经元。