Horseradish peroxidase cDNA as a marker for electron microscopy in neurons
Horseradish peroxidase cDNA as a marker for electron microscopy in neurons
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DOI：
10.1016/j.jneumeth.2007.06.004
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发表时间：
2007-09-30
影响因子：
3
通讯作者：
Hu, Yunming
中科院分区：
文献类型：
--
作者：
Schikorski, Thomas;Young, Samuel M., Jr.;Hu, Yunming
电子显微镜缺乏可靠且方便的神经元基因转移标记。为了促进分子遗传学方法在此类研究中的使用，我们引入了使用辣根过氧化物酶 (HRP) cDNA 作为标记物，用于识别转染的神经元，用于相关光学和电子显微镜 (EM)。通过在 cDNA 侧翼添加内质网 (ER) 保留信号，表达的 HRP 被定位并保留在神经元的 ER 内。该构建体置于突触蛋白启动子的控制下，导致 HRP 的神经元特异性表达。在培养物中转染海马神经元并在固定后用二氨基联苯胺 (DAB) 对 HRP 进行可视化后，HRP 阳性神经元的细胞体和近端树突都可以通过光学显微镜轻松识别。经过 EM 处理后，重度 DAB 标记的 ER 在转染神经元的细胞体和树突中都很明显。此外，远端树突、轴突和突触的标记也很明显。转染神经元的结构保存是最佳的，这对于戊二醛固定组织来说是典型的。由于 ER 保留信号，HRP 包含在 ER 内部，并且所有其他组件的超微结构没有被遮挡并且可以定量获取。这些特性使得 HRP 共转染成为细胞生物学和神经科学领域现代电子显微镜的潜在强大工具。 (c) 2007 Elsevier B.V. 保留所有权利。
A reliable and convenient marker of gene transfer in neurons is lacking for electron microscopy. To facilitate the use of molecular genetic approaches in such studies, we introduce the use of horseradish peroxidase (HRP) cDNA as a marker that identifies transfected neurons for correlated light and electron microscopy (EM). By flanking the cDNA with an endoplasmic reticulum (ER)-retention signal, expressed HRP was localized and retained inside the ER of neurons. The construct was placed under the control of the synapsin promoter resulting in neuron-specific expression of HRP. After transfection of hippocampal neurons in culture and visualization of HRP with diaminobenzidine (DAB) after fixation, both cell bodies and proximal dendrites of HRP-positive neurons were readily identifiable by light microscopy. After processing for EM, the heavily DAB-labeled ER was obvious in both cell bodies and dendrites of transfected neurons. In addition, the labeling of distal dendrites, axons, and synapses was apparent. The structural preservation of transfected neurons was optimal as is typical for glutaraldehyde-fixed tissue. Due to the ER-retention signal, HRP was contained inside the ER and the ultrastructure of all other components was not occluded and was quantitatively accessible. These characteristics make HRP co-transfection a potentially powerful tool for modem electron microscopy in both cell biology and neuroscience. (c) 2007 Elsevier B.V. All rights reserved.