A facile method for fluorescent visualization of newly synthesized fibrous collagen by capturing the allysine aldehyde groups serving as cross-link precursors

The fibrous structures of collagen provide physical strength and stability to tissues and organs. Abnormalities in their orientation, growth, and remodeling cause morphogenetic defects and diseases including fibrosis, highlighting the importance of understanding how collagen fibers are organized within tissues. However, this process remains difficult to study, as methods for fluorescently labeling collagen fibers with simple protocols and visualizing their three-dimensional structure are still limited. Here we describe a convenient method for fluorescent labeling of collagen fibers in vertebrate tissues. Using DAF-FM, a probe originally developed for nitric oxide detection, premature collagen fibers can be visualized through covalent binding to allysine residues, which serve as precursors of collagen cross-linking. We further show that combining two probes with different emission spectra, DAF-FM and DAR-4M, enables pulse-chase labeling of newly synthesized collagen fibers. This approach provides a practical tool for investigating collagen dynamics during tissue development and remodeling.