TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability
Abstract 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,...
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| Format: | Article |
| Language: | English |
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BMC
2023-05-01
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| Series: | Cell & Bioscience |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13578-023-01043-1 |
| _version_ | 1827939734793486336 |
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| author | Kang Wang Yuxin Yu Yinhui Xu Yingzi Yue Fang Zhao Wenyang Feng Yijie Duan Weicheng Duan Jingjing Yue Zhiyun Liao Peng Fei Hui Sun Bo Xiong |
| author_facet | Kang Wang Yuxin Yu Yinhui Xu Yingzi Yue Fang Zhao Wenyang Feng Yijie Duan Weicheng Duan Jingjing Yue Zhiyun Liao Peng Fei Hui Sun Bo Xiong |
| author_sort | Kang Wang |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-03-13T08:58:51Z |
| format | Article |
| id | doaj.art-d9ce4f9e6276438bbeed05d42dc8e089 |
| institution | Directory Open Access Journal |
| issn | 2045-3701 |
| language | English |
| last_indexed | 2024-03-13T08:58:51Z |
| publishDate | 2023-05-01 |
| publisher | BMC |
| record_format | Article |
| series | Cell & Bioscience |
| spelling | doaj.art-d9ce4f9e6276438bbeed05d42dc8e0892023-05-28T11:28:43ZengBMCCell & Bioscience2045-37012023-05-0113111510.1186/s13578-023-01043-1TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stabilityKang Wang0Yuxin Yu1Yinhui Xu2Yingzi Yue3Fang Zhao4Wenyang Feng5Yijie Duan6Weicheng Duan7Jingjing Yue8Zhiyun Liao9Peng Fei10Hui Sun11Bo Xiong12Department of Forensic Medicine, Tongji Medical College, Huazhong University of Science and TechnologyDepartment of Endocrinology, Union Hospital, Tongji Medical College, Huazhong University of Science and TechnologyDepartment of Pediatric Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and TechnologyKey Laboratory of Environment and Health (HUST), Ministry of Education, School of Public Health, Tongji Medical College, Huazhong University of Science and TechnologySchool of Optical and Electronic Information- Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and TechnologySchool of Optical and Electronic Information- Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and TechnologyDepartment of Forensic Medicine, Tongji Medical College, Huazhong University of Science and TechnologyDepartment of Forensic Medicine, Tongji Medical College, Huazhong University of Science and TechnologyCollege of Engineering, Huazhong Agricultural UniversityCancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and TechnologySchool of Optical and Electronic Information- Wuhan National Laboratory for Optoelectronics, Huazhong University of Science and TechnologyDepartment of Endocrinology, Union Hospital, Tongji Medical College, Huazhong University of Science and TechnologyDepartment of Forensic Medicine, Tongji Medical College, Huazhong University of Science and TechnologyAbstract 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.https://doi.org/10.1186/s13578-023-01043-1PACTCLARITYZebrafishImmunofluorescenceTyramide signal amplificationTissue clearing |
| spellingShingle | Kang Wang Yuxin Yu Yinhui Xu Yingzi Yue Fang Zhao Wenyang Feng Yijie Duan Weicheng Duan Jingjing Yue Zhiyun Liao Peng Fei Hui Sun Bo Xiong TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability Cell & Bioscience PACT CLARITY Zebrafish Immunofluorescence Tyramide signal amplification Tissue clearing |
| title | TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability |
| title_full | TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability |
| title_fullStr | TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability |
| title_full_unstemmed | TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability |
| title_short | TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability |
| title_sort | tsa pact a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity specificity and stability |
| topic | PACT CLARITY Zebrafish Immunofluorescence Tyramide signal amplification Tissue clearing |
| url | https://doi.org/10.1186/s13578-023-01043-1 |
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