Proliferation studies in the chick ear‐forming region using EdU labeling

Abstract

Cell proliferation studies are an important experimental tool, which commonly include PCNA and phospho‐histone H3 antibodies again proteins present in mitotic cells, and the thymidine analogues tritiated thymidine and BrdU, which label cells during S‐phase. Both the latter methods have significant drawbacks, with low sensitivity in the case of tritiated thymidine and a denaturation step during BrdU detection that destroys most cellular epitopes, requiring careful optimization. The BrdU antibody is also large and tissue penetration can be difficult, moreover multiplex antibody detection is compromised due to the denaturation step. EdU is a closely chemically related molecule to BrdU. Detection is achieved by a copper catalyzed reaction requiring a small fluorescently conjugated azide. 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 fluorescently labeled tissues. To understand the role of proliferation in ear formation we are undertaking proliferation studies in the ear‐forming region of early chick embryos.