Optimization of immunolabeling and clearing techniques for indelibly labeled memory traces.
Optimization of immunolabeling and clearing techniques for indelibly labeled memory traces.
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DOI：
10.1002/hipo.22951
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发表时间：
2018-07
期刊：
影响因子：
3.5
通讯作者：
Denny CA
中科院分区：
文献类型：
--
作者：
Pavlova IP;Shipley SC;Lanio M;Hen R;Denny CA
最近的遗传工具使研究人员能够可视化和操纵大脑小区域的记忆痕迹（即印迹）。然而，最终目标是可视化整个大脑的记忆痕迹，以便更好地理解记忆如何存储在神经网络中以及多个记忆如何共存。完整的组织透明化和成像是一个新的、快速增长的焦点领域，可以完成这项任务。在这里，我们利用了领先的全脑清除方案，并将其应用于 ArcCreERT2 小鼠，这是一种能够对记忆痕迹进行不可磨灭标记的小鼠品系。我们发现 CLARITY 和 PACT 极大地扭曲了组织，iDISCO 猝灭了增强型黄色荧光蛋白 (EYFP) 荧光并阻碍了免疫标记。替代的透明解决方案，例如叔丁醇，避免了这些有害影响，但仍然不允许全脑免疫标记。 CUBIC 和带有试剂 1A 的 CUBIC 产生了改进的抗体渗透并保留了 EYFP 荧光，但也不允许全脑记忆轨迹可视化。用 Reagent-1A 修饰 CUBIC 导致大脑深部区域的 EYFP 荧光保留和立即早期基因 (IEG) Arc 的免疫标记；然而，优化的记忆痕迹标记仍然需要将组织切成毫米厚的组织切片。总之，我们的数据表明，使用 Reagent-1A* 的 CUBIC 是可重复清除和免疫标记的理想方法，用于可视化 ArcCreERT2 小鼠毫米厚组织切片中的记忆痕迹。
Recent genetic tools have allowed researchers to visualize and manipulate memory traces (i.e. engrams) in small brain regions. However, the ultimate goal is to visualize memory traces across the entire brain in order to better understand how memories are stored in neural networks and how multiple memories may coexist. Intact tissue clearing and imaging is a new and rapidly growing area of focus that could accomplish this task. Here, we utilized the leading protocols for whole-brain clearing and applied them to the ArcCreERT2 mice, a murine line that allows for the indelible labeling of memory traces. We found that CLARITY and PACT greatly distorted the tissue, and iDISCO quenched enhanced yellow fluorescent protein (EYFP) fluorescence and hindered immunolabeling. Alternative clearing solutions, such as tert-Butanol, circumvented these harmful effects, but still did not permit whole-brain immunolabeling. CUBIC and CUBIC with Reagent 1A produced improved antibody penetration and preserved EYFP fluorescence, but also did not allow for whole-brain memory trace visualization. Modification of CUBIC with Reagent-1A resulted in EYFP fluorescence preservation and immunolabeling of the immediate early gene (IEG) Arc in deep brain areas; however, optimized memory trace labeling still required tissue slicing into mm-thick tissue sections. In summary, our data show that CUBIC with Reagent-1A* is the ideal method for reproducible clearing and immunolabeling for the visualization of memory traces in mm-thick tissue sections from ArcCreERT2 mice.
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影响因子：
25
作者：
通讯作者：
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影响因子：
16.2
作者：
Denny CA;Kheirbek MA;Alba EL;Tanaka KF;Brachman RA;Laughman KB;Tomm NK;Turi GF;Losonczy A;Hen R
通讯作者：
Hen R
影响因子：
48
作者：
Pan, Chenchen;Cai, Ruiyao;Ertuerk, Ali
通讯作者：
Ertuerk, Ali
影响因子：
8.8
作者：
Belle, Morgane;Godefroy, David;Chedotal, Alain
通讯作者：
Chedotal, Alain
影响因子：
14.8
作者：
通讯作者：
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