On May 26, 2023, XIONG Bo 's research group from Department of Forensic Medicine, Tongji Medical College, Huazhong University of Science and Technology and SUN Hui 's research group from Department of Endocrinology, Union Hospital collaborated on Cell & Bioscience ( IF = 9.584 ), the official academic journal of the American Association of Chinese Biologists, and published an online paper entitled TSA-PACT : a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity. Research paper on specificity and stability.

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.

It is worth mentioning that the development of this project began with the undergraduate innovation project ' Establishment and Application of Zebrafish Tissue Transparency Experimental System Based on PACT Technology '. At the beginning of 2019, YU Yuxin and XU Yinhui, grade 3 students of 8-year clinical medicine major, applied for and approved the relevant innovation projects. After 1 year of research and training, the project has made initial progress and successfully concluded. However, there were still many technical details that needed to be further improved. In the following three years, with the help of doctoral student WANG Kang, under the guidance of Professor XIONG Bo and Professor SUN Hui, the two students continued to use their spare time to repeatedly study and continuously optimize the experimental process, and finally achieved good experimental results. Then, YU Yuxin and XU Yinhui participated in the whole process of data collation, paper writing, submission, supplementary experiment, rework and other processes, obtained sufficient scientific research training, and achieved the goal of publishing the results of the innovation project in academic journals. Professor XIONG Bo and Professor SUN Hui of Huazhong University of Science and Technology are the co-communicating authors of this paper. Dr. WANG Kang of the Department of Forensic Medicine ( now a full-time teacher of Nanjing Medical University ), YU Yuxin and XU Yinhui of the 8-year clinical medicine major are the co-first authors of this paper. The research is supported by the National Natural Science Foundation of China, the Technology Innovation Fund of the Innovation Research Institute of Huazhong University of Science and Technology and the Natural Science Research Fund of Jiangsu Province.

Paper link：TSA-PACT: a method for tissue clearing and immunofluorescence staining on zebrafish brain with improved sensitivity, specificity and stability – DOAJ