Immunocytochemistry and quantification of protein colocalization in cultured neurons
Immunocytochemistry and quantification of protein colocalization in cultured neurons
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DOI：
10.1038/nprot.2006.220
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
McAllister, A. Kimberley
中科院分区：
文献类型：
--
作者：
Glynn, Marian W.;McAllister, A. Kimberley
该协议详细介绍了一种量化神经元培养物中蛋白质分布和共定位的方法。为此，该协议包括执行免疫细胞化学、采集荧光图像和量化多通道荧光图像的详细步骤和注意事项。定量免疫染色神经元的成功取决于目标蛋白质的可及性、抗体的敏感性和特异性、收集图像的信噪比以及定量方法的敏感性。与其他常用的量化方法相比，此处详细介绍的协议需要手动选择点并减去为每个神经突选择的背景。这种方法可靠且独特，可以检测低信噪比图像中的蛋白质，这是发育中神经元的特征。因此，该方法在图像分析中占有重要地位，而其他已发表的方法却很难解决这一问题。一般来说，免疫细胞化学需要3.5-7小时，1.5小时内即可对1个三重免疫染色的神经元进行定量。
This protocol details a method to quantify the distribution of protein and colocalization in neuronal cultures. To that end, this protocol includes itemized steps and considerations for performing immunocytochemistry, acquiring fluorescence images and quantifying multichannel fluorescence images. Success in quantifying immunostained neurons relies on the accessibility of the proteins of interest, the sensitivity and specificity of antibodies, the signal-to-noise ratio of the collected image and the sensitivity of the quantification method. In contrast to other commonly employed methods for quantification, the protocol detailed here requires manual selection of punctae and subtraction of background selected for each neurite. This approach reliably and uniquely allows for detection of proteins in low signal-to-noise ratio images, which are characteristic of developing neurons. Thus, this method serves an important niche in image analysis poorly addressed by alternative published methods. In general, immunocytochemistry requires 3.5-7 h, and one triple-immunostained neuron can be quantified in 1.5 h.