KneEZ Clear: an Effective Tissue Clearing Method for Intact Mouse Joints, including the Knee v2
Year: 2024
Type: preprint
Abstract: KneEZ Clear is a modified version of the EZ Clear method (Hsu et al., 2022), optimized for clearing mouse musculoskeletal tissues. KneEZ Clear works well for the clearing of mouse knee and ankle joints, spines, temporomandibular joints, and teeth. Critical factors that impact the tissue’s optical transparency include fixation and decalcification time and temperature, as well as gradual lipid removal. This protocol includes injection with fluorescently conjugated lectin to label the vasculature, followed by collection of tissue samples, fixation, tissue delipidation, and refractive index matching (e.g. tissue clearing). While lectin-labeled vasculature serves as a good positive control, it can be omitted when one only wants to look at endogenous fluorescence. In this case, proceed directly to transcardial perfusion of PBS and fixative. To efficiently label the microvasculature and venous endothelium, we employ a combinatorial approach of retroorbital perfusion followed by transcardial perfusion pre-fixation, followed by an additional pulse of diluted lectin post-fixation. This combination results in superior fluorescent labeling of the venous and capillary vasculature, as well as larger diameter arterial and venous vessels.
Institutions: Baylor College of Medicine, University of Virginia
Language: English
Subfield: Biophysics
Domain: Life Sciences
Open Access status: gold
OpenAlex ID: W4411796594