Details

Autor(en) / Beteiligte

• Fischer, Adrian
• Correa-Gallegos, Donovan
• Wannemacher, Juliane
• Christ, Simon
• Machens, Hans-Günther
• Rinkevich, Yuval

Titel

In vivo fluorescent labeling and tracking of extracellular matrix

Ist Teil von

• Nature protocols, 2023-10, Vol.18 (10), p.2876-2890

Ort / Verlag

London: Nature Publishing Group UK

Erscheinungsjahr

2023

Beschreibungen/Notizen

• Connective tissues are essential building blocks for organ development, repair and regeneration. However, we are at the early stages of understanding connective tissue dynamics. Here, we detail a method that enables in vivo fate mapping of organ extracellular matrix (ECM) by taking advantage of a crosslinking chemical reaction between amine groups and N -hydroxysuccinimide esters. This methodology enables robust labeling of ECM proteins, which complement previous affinity-based single-protein methods. This protocol is intended for entry-level scientists and the labeling step takes between 5 and 10 min. ECM ‘tagging’ with fluorophores using N -hydroxysuccinimide esters enables visualization of ECM spatial modifications and is particularly useful to study connective tissue dynamics in organ fibrosis, tumor stroma formation, wound healing and regeneration. This in vivo chemical fate mapping methodology is highly versatile, regardless of the tissue/organ system, and complements cellular fate-mapping techniques. Furthermore, as the basic chemistry of proteins is highly conserved between species, this method is also suitable for cross-species comparative studies of ECM dynamics. Key points This protocol describes a method for broad-spectrum, in vivo fluorescent labeling and tracking of extracellular matrix (ECM) proteins through the systemic or local application of N -hydroxysuccinimide esters. This flexible approach can be adapted to a variety of organ systems and wounding models. In contrast to common ECM-tracing methods that enable labeling single ECM proteins, this N -hydroxysuccinimide ester-based chemical technique tags virtually all ECM proteins to unveil more general ECM mechanics. A protocol for broad-spectrum, in vivo fluorescent labeling and tracking of extracellular matrix proteins through the systemic or local application of N -hydroxysuccinimide esters. This flexible approach can be adapted for a variety of organ systems and wounding models.