5-Ethynyl-2′-Deoxyuridine Labeling Detects Proliferating Cells in the Regenerating Avian Cochlea

Objectives/Hypothesis: The avian cochlea regenerates hair cells following aminoglycoside treatment through supporting cell proliferation. Immunocytochemical labeling of 5-bromo-2'-deoxyuridine (BrdU), a thymidine analog, is a popular nonradioactive marker for identifying cells in the DNA synthesis (S phase) of the cell cycle. However, it requires harsh treatments to denature double-stranded DNA for the antibody to bind BrdU. We explored a new method using 5-ethynyl-2'-deoxyuridine (EdU) as a thymidine analog and a nonantibody azide/alkyne reaction between EdU and the fluorescent probe. We propose that EdU is as effective as BrdU, but without the requirement for harsh denaturation or the use of antibodies for detection. Study Design: Two-week-old chicks received a single gentamicin injection followed by a single EdU injection 72 hours later. Cochleae were extracted 4-8 hours later, fixed, and processed for fluorescent detection of EdU. Methods: Cochleae were processed for detection of incorporated EdU using the Click-iT Imaging Kit (Invitrogen/Molecular Probes, Carlsbad, CA) and colabeled with Sox2, myosin VI, or myosin VIIa antibodies. Whole-mount cochlear preparations were examined with confocal microscopy. Results: Supporting cells incorporated EdU into their newly synthesized DNA during the 4-8 hours following the EdU injection and were readily detected with little background signal. The intensity and quantity of cells labeled were similar to or better than that seen for BrdU. Conclusions: The EdU method is as effective as BrdU, without requiring harsh denaturation or secondary antibodies to identify proliferating cells. Thus, the nonantibody EdU system allows more flexibility by enabling colabeling with multiple antibodies to other cellular proteins involved in regeneration.