TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability.
TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability.
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DOI：
10.1186/s13578-023-01043-1
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发表时间：
2023-05-26
影响因子：
7.5
通讯作者：
Xiong, Bo
中科院分区：
文献类型：
--
作者：
Wang, Kang;Yu, Yuxin;Xu, Yinhui;Yue, Yingzi;Zhao, Fang;Feng, Wenyang;Duan, Yijie;Duan, Weicheng;Yue, Jingjing;Liao, Zhiyun;Fei, Peng;Sun, Hui;Xiong, Bo
关键词：
对于脑结构和功能的全面研究，全脑的荧光成像是必不可少的。它需要细胞或分子分辨率的大规模体积成像，这可能是相当具有挑战性的。组织透明化技术的最新进展（例如，PACT、PLCITY）通过改变样品的折射率以产生透明度来提供新的解决方案。然而，它一直难以获得高质量的结果，通过免疫荧光（IF）染色的澄清样品。为了解决这个问题，我们开发了TSA-PACT，一种结合酪胺信号放大（TSA）和PACT的方法，将样品转化为组装有共价荧光生物标志物的水凝胶聚合框架。我们表明，TSA-PACT能够将斑马鱼大脑的不透明度降低90%以上，并保持良好的结构。与传统方法相比，TSA-PACT实现了约10倍的信号放大和2倍的信噪比改善。此外，该结构和荧光信号均保持至少16个月，具有优异的信号保留率。总体而言，该方法提高了斑马鱼幼鱼和成年鱼全脑免疫荧光信号的灵敏度、特异性和稳定性，适用于精细结构分析、神经回路作图和三维细胞计数。在线版本包含补充材料，可通过10.1186/s13578-023-01043-1获得。
For comprehensive studies of the brain structure and function, fluorescence imaging of the whole brain is essential. It requires large-scale volumetric imaging in cellular or molecular resolution, which could be quite challenging. Recent advances in tissue clearing technology (e.g. CLARITY, PACT) provide new solutions by homogenizing the refractive index of the samples to create transparency. However, it has been difficult to acquire high quality results through immunofluorescence (IF) staining on the cleared samples. To address this issue, we developed TSA-PACT, a method combining tyramide signal amplification (TSA) and PACT, to transform samples into hydrogel polymerization frameworks with covalent fluorescent biomarkers assembled. We show that TSA-PACT is able to reduce the opacity of the zebrafish brain by more than 90% with well-preserved structure. Compared to traditional method, TSA-PACT achieves approximately tenfold signal amplification and twofold improvement in signal-to-noise ratio (SNR). Moreover, both the structure and the fluorescent signal persist for at least 16 months with excellent signal retention ratio. Overall, this method improves immunofluorescence signal sensitivity, specificity and stability in the whole brain of juvenile and adult zebrafish, which is applicable for fine structural analysis, neural circuit mapping and three-dimensional cell counting. The online version contains supplementary material available at 10.1186/s13578-023-01043-1.
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