Protocol to study presynaptic protein synthesis in ex vivo mouse hippocampal slices using HaloTag technology.
Protocol to study presynaptic protein synthesis in ex vivo mouse hippocampal slices using HaloTag technology.
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DOI：
10.1016/j.xpro.2022.101986
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发表时间：
2023-03-17
期刊：
影响因子：
--
通讯作者：
Castillo, Pablo E.
中科院分区：
文献类型：
--
作者：
Kharod, Shivani C.;Monday, Hannah R.;Yoon, Young J.;Castillo, Pablo E.
哺乳动物大脑中的突触前钮扣通常很小，难以操作和研究。在这里，我们提出了一种应用HaloTag自标记技术检测急性小鼠海马切片中完整的突触前苔藓纤维扣的局部蛋白质合成的方案。我们描述了将表达halotag的病毒立体定向注射到感兴趣的大脑区域，然后进行脑切片制备。然后我们详细介绍了halotag融合蛋白的标记和图像采集，以在完整的电路中可视化标记的蛋白质。有关该协议的使用和执行的完整细节，请参阅Monday等人（2022）。立体定向注入感兴趣的halottag融合基因到感兴趣的脑区制备成年小鼠急性海马切片（P35-45）。光晕-肌动蛋白标记检测苔藓纤维钮孔中局部新合成的肌动蛋白成像和脑切片图像处理出版商注：开展任何实验方案都需要遵守当地机构的实验室安全和伦理指导方针。哺乳动物大脑中的突触前钮扣通常很小，难以操作和研究。在这里，我们提出了一种应用Halotag自标记技术检测急性小鼠海马切片中完整的突触前苔藓纤维扣的局部蛋白质合成的方案。我们描述了立体定向注射表达halottag的病毒进入感兴趣的大脑区域，然后是脑切片制备。然后详细介绍了halottag融合蛋白的标记和图像采集，以在完整的电路中可视化标记的蛋白质。
Presynaptic boutons in the mammalian brain are typically small and difficult to manipulate and study. Here, we present a protocol applying HaloTag self-labeling technology to detect de novo local protein synthesis in intact presynaptic mossy fiber boutons from acute mouse hippocampal slices. We describe stereotaxic injection of HaloTag-expressing virus into the brain region of interest, followed by brain slice preparation. We then detail the labeling of HaloTag-fused protein and image acquisition to visualize the labeled protein in an intact circuit. For complete details on the use and execution of this protocol, please refer to Monday et al. (2022). Stereotaxic injection of Halotag-fused gene of interest into brain region of interest Preparation of acute hippocampal slices from adult mice (P35-45). Halo-actin labeling to detect local newly synthesized actin in mossy fiber boutons Imaging and Airyscan processing of brain slice images Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Presynaptic boutons in the mammalian brain are typically small and difficult to manipulate and study. Here, we present a protocol applying Halotag self-labeling technology to detect de novo local protein synthesis in intact presynaptic mossy fiber boutons from acute mouse hippocampal slices. We describe stereotaxic injection of Halotag-expressing virus into the brain region of interest, followed by brain slice preparation. We then detail the labeling of Halotag-fused protein and image acquisition to visualize the labeled protein in an intact circuit.
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影响因子：
3.7
作者：
Schmidt PM;Sparrow LG;Attwood RM;Xiao X;Adams TE;McKimm-Breschkin JL
通讯作者：
McKimm-Breschkin JL
影响因子：
3.4
作者：
Hein, Birka;Willig, Katrin I.;Hell, Stefan W.
通讯作者：
Hell, Stefan W.
DOI：
10.1073/pnas.1614267113
发表时间：
2016-11-01
影响因子：
11.1
作者：
Yoon, Young J.;Wu, Bin;Singer, Robert H.
通讯作者：
Singer, Robert H.
Halotag技术：用于生物医学应用的多功能平台。
影响因子：
4.7
作者：
England, Christopher G.;Luo, Haiming;Cai, Weibo
通讯作者：
Cai, Weibo
影响因子：
16.2
作者：
Galimberti, Ivan;Gogolla, Nadine;Caroni, Pico
通讯作者：
Caroni, Pico