Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics
Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics
Abstract Various small molecules have been used as functional probes for tissue imaging in medical diagnosis and pharmaceutical drugs for disease treatment. The spatial distribution, target selectivity, and diffusion/extrusion kinetics of small molecules in structurally complicated specimens are critical for function. However, robust methods for precisely evaluating these parameters in the brain have been limited. Herein we report a new method termed “Fixation-driven chemical crosslinking of exogenous ligands (FixEL)” which traps and images exogenously administered molecules-of-interest (MOI) in complex tissues. This method relies on proteins-MOI interactions, and chemical crosslinking of amine-tethered MOI with paraformaldehyde used for perfusion fixation. FixEL is used to obtain images of the distribution of the small molecules and their dynamics, which addresses selective/nonselective binding to proteins, time-dependent localization changes, and diffusion/retention kinetics of MOI such as PET tracer derivatives or drug-like small molecules. Clear imaging of a nanobody distributed in the whole brain was also achieved with high spatial resolution using 2D/3D mode.
Tsushima Akihiro、Tamura Tomonori、Aricescu A. Radu、Hamachi Itaru、Ishikawa Mamoru、Oh Jae Hoon、Nonaka Hiroshi、Mino Takeharu、Amaike Kazuma、Sakamoto Seiji、Kiyonaka Shigeki、Yuzaki Michisuke、Watanabe Yu、Kakegawa Wataru、Miura Eriko
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University||ERATO (Exploratory Research for Advanced Technology, JST)Division of Structural Biology, University of Oxford||Neurobiology Division, MRC Laboratory of Molecular BiologyDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University||ERATO (Exploratory Research for Advanced Technology, JST)ERATO (Exploratory Research for Advanced Technology, JST)ERATO (Exploratory Research for Advanced Technology, JST)Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University||ERATO (Exploratory Research for Advanced Technology, JST)Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityERATO (Exploratory Research for Advanced Technology, JST)||Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya UniversityDepartment of Neurophysiology, Keio University School of MedicineDepartment of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto UniversityERATO (Exploratory Research for Advanced Technology, JST)||Department of Neurophysiology, Keio University School of MedicineDepartment of Neurophysiology, Keio University School of Medicine
医学研究方法基础医学生物科学研究方法、生物科学研究技术
Tsushima Akihiro,Tamura Tomonori,Aricescu A. Radu,Hamachi Itaru,Ishikawa Mamoru,Oh Jae Hoon,Nonaka Hiroshi,Mino Takeharu,Amaike Kazuma,Sakamoto Seiji,Kiyonaka Shigeki,Yuzaki Michisuke,Watanabe Yu,Kakegawa Wataru,Miura Eriko.Revisiting PFA-mediated tissue fixation chemistry: FixEL enables trapping of small molecules in the brain to visualize their distribution dynamics[EB/OL].(2025-03-28)[2025-05-08].https://www.biorxiv.org/content/10.1101/2021.12.21.473647.点此复制
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