Chick embryo proliferation studies using EdU labeling

Abstract

Cell proliferation studies are an important experimental tool. The most commonly used thymidine analogues, tritiated thymidine and bromodeoxyuridine (BrdU) label cells during S-phase. Both methods have significant drawbacks: low sensitivity in the case of tritiated thymidine and a denaturation step during BrdU detection that destroys most cellular epitopes, requiring careful optimization. The antibody against BrdU is also large and tissue penetration can be difficult. EdU (5'-ethynyl-2'-deoxyuridine) is closely chemically related to BrdU, with detection achieved by a copper catalyzed reaction requiring a small fluorescently conjugated azide. Cell cultures, flow cytometry and high throughput studies using EdU-labeled cells is exceptionally fast and does not require denaturation or antibodies. We have developed a tissue-labeling technique in chick embryos using EdU. Following EdU chemistry to detect proliferating cells, the tissue can undergo immunolabeling. We demonstrate fluorescent EdU chemistry followed by Tuj1 antibody staining resulting in multiplex fluorescent tissues.