大鼠皮质神经元中丝状肌动蛋白（F-肌动蛋白）斑点的定量分析。
Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons.
作者信息
Li Hailong, Aksenova Marina, Bertrand Sarah J, Mactutus Charles F, Booze Rosemarie
机构信息
Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina.
Laboratory Program in Behavioral, Neuroscience, Department of Psychology, University of South Carolina;
出版信息
J Vis Exp. 2016 Feb 10(108):e53697. doi: 10.3791/53697.
Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 µm) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.
丝状肌动蛋白（F-肌动蛋白）在轴突发生、突触可塑性和突触稳定性中起主要作用。富含树突状F-肌动蛋白的结构变化表明突触完整性和连接性发生改变。在此，我们提供了一个详细的方案，用于培养原代大鼠皮质神经元、用鬼笔环肽染色检测F-肌动蛋白斑点以及后续的定量技术。首先，将E18大鼠胚胎的额叶皮质解离成低密度细胞培养物，然后将神经元在体外培养至少12 - 14天。经过实验处理后，用AlexaFluor 488鬼笔环肽（标记树突状F-肌动蛋白斑点）和微管相关蛋白2（MAP2；验证神经元细胞和树突完整性）对皮质神经元进行染色。最后，使用专门的软件分析和定量随机选择的神经元树突。在具有连续MAP2免疫荧光的二级树突分支（长度范围25 - 75 µm）上识别富含F-肌动蛋白的结构。这里介绍的方案将是研究实验处理后树突突触结构变化的一种有用方法。
Zotero 插件