EdU-Based Assay of Cell Proliferation and Stem Cell Quiescence in Skeletal Tissue Sections

Abstract

Identifying and tracking proliferating and quiescent cells in situ is an important phenotyping component of skeletal tissues in development, physiology and disease. Among all the methods that exist, which include immunostaining for cell cycle-specific proteins, the gold standards use thymidine analogs. These compounds label proliferating cells by being incorporated into de novo–synthesized genomic DNA. 5-bromo-2′-deoxyuridine (BrdU) has traditionally been used for this purpose, but its detection is lengthy and requires harsh treatment of tissue sections to give access of anti-BrdU antibody to DNA. An alternative, more recently developed, uses 5-ethynyl-2′-deoxyuridine (EdU). This thymidine analog is detected by click chemistry, that is, covalent cross-linking of its ethynyl group with a fluorescent azide that is small enough to easily penetrate native tissues and reach DNA. In addition to being simple and quick, this EdU-based assay is compatible with other protocols, such as immunostaining, on the same tissue sections. We here describe an EdU-based protocol optimized to label and functionally assess actively proliferating cells as well as slowly dividing cells, including stem cells, in mouse skeletal tissues.