PUBLICATION
TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability
- Authors
- Wang, K., Yu, Y., Xu, Y., Yue, Y., Zhao, F., Feng, W., Duan, Y., Duan, W., Yue, J., Liao, Z., Fei, P., Sun, H., Xiong, B.
- ID
- ZDB-PUB-230528-36
- Date
- 2023
- Source
- Cell & Bioscience 13: 97 (Journal)
- Registered Authors
- Keywords
- Brain damage, Brain imaging, CLARITY, Immunofluorescence, PACT, Tissue clearing, Tyramide signal amplification, Zebrafish
- MeSH Terms
- none
- PubMed
- 37237300 Full text @ Cell Biosci.
Citation
Wang, K., Yu, Y., Xu, Y., Yue, Y., Zhao, F., Feng, W., Duan, Y., Duan, W., Yue, J., Liao, Z., Fei, P., Sun, H., Xiong, B. (2023) TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability. Cell & Bioscience. 13:97.
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.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping