Helmholtz Zentrum München, Institute of Regenerative Biology & Medicine, Munich, Germany.
Technical University of Munich, School of Medicine, Klinikum rechts der Isar, Department of Plastic and Hand Surgery, Munich, Germany.
Nat Protoc. 2023 Oct;18(10):2876-2890. doi: 10.1038/s41596-023-00867-y. Epub 2023 Aug 9.
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.
结缔组织是器官发育、修复和再生的重要组成部分。然而，我们对结缔组织动态的了解还处于早期阶段。在这里，我们详细介绍了一种方法，该方法利用胺基与 N-羟基琥珀酰亚胺酯之间的交联化学反应，实现了器官细胞外基质 (ECM) 的体内命运图谱。这种方法能够对 ECM 蛋白进行稳健标记，这补充了以前基于亲和力的单蛋白方法。本方案适用于入门级科学家，标记步骤需要 5 到 10 分钟。使用 N-羟基琥珀酰亚胺酯对 ECM 进行荧光标记，可实现 ECM 空间修饰的可视化，特别适用于研究器官纤维化、肿瘤基质形成、伤口愈合和再生中的结缔组织动态。这种体内化学命运图谱方法具有高度通用性，与组织/器官系统无关，并补充了细胞命运图谱技术。此外，由于蛋白质的基本化学性质在物种间高度保守，因此该方法也适用于 ECM 动态的跨物种比较研究。
