Tissue clearing and three-dimensional imaging of intact tissues: a review on FACT protocol
Farrar Z. Afshar A. Zare A. Mussin N.M. Kaliyev A.A. Zhilisbayeva K.R. Mahdipour M. Tamadon A.
2024Taylor and Francis Ltd.
Journal of Histotechnology
2024#47Issue 3126 - 142 pp.
FACT is a developed technique for clearing tissues that does not use acrylamide. Since the removal of lipids is crucial for transparency and efficient antibody staining throughout the tissue, especially for microscopy and imaging, it is a harmful process that can cause the loss of important biological molecules such as proteins. The FACT technique overcomes this by chemically bonding the membrane and intracellular proteins with the extracellular matrix, creating a massive 3D hydrogel matrix and providing structural support to fortify the tissue during processing. Compared to other acrylamide-based techniques, the FACT technique requires less labor and harmful chemicals and is therefore considered a more suitable option. In this study, we describe the complete FACT protocol for antibody staining and imaging of whole-cleared tissues while preserving structure and improving image quality. The protocol includes tissue perfusion, fixation, clearing, antibody staining, refractive index matching (RIM) (), microscopy, and imaging. The timing for each step varies depending on the size, weight, and type of tissue, as well as the type of immunostaining. We provide an example of the FACT protocol using mouse brain tissue, which demonstrates its suitability for molecular interrogation analysis of large tissues. The FACT technique has been successfully performed on different types of tissues, making it a favorable choice for a variety of applications.
confocal , Imaging , microscopy , three-dimensional , tissues
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Student Research Committee, Bushehr University of Medical Sciences, Bushehr, Iran
Department of Research and Development, PerciaVista R&D Co., Shiraz, Iran
Department of General Surgery, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
Department of Scientific Work, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran
Department of Natural Sciences, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan
Student Research Committee
Department of Research and Development
Department of General Surgery
Department of Scientific Work
Stem Cell Research Center
Department of Applied Cell Sciences
Department of Natural Sciences
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