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BrdU assay for neurogenesis in rodents

Nature Protocols volume 1, pages 1399–1405 (2006)Cite this article

Abstract

Neurogenesis within the adult central nervous system is demonstrated using an exogenous cell tracer, 5'-bromo-2'-deoxyuridine (BrdU), in combination with endogenous neuronal markers. Specific primary antibodies raised against these markers are widely available and their visualization is possible with the use of fluorescently tagged secondary antibodies. BrdU is a thymidine analog that incorporates into dividing cells during DNA synthesis. Once incorporated into the new DNA, BrdU will remain in place and be passed down to daughter cells following division. Typically, BrdU is injected intraperitoneally. Different survival times required by the desired experimental time-line will yield data on specific phases of neurogenesis: proliferation, differentiation and maturation. One of the drawbacks of using BrdU is the dependence on a stressful injection procedure and uncertain penetration of the targeted cells with a uniform concentration of the compound. Thus, for experiments requiring measurements of cell proliferation, Ki67 can be used as an acceptable alternative. The protocol takes 3–5 d, allowing for sectioning and staining.

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Figure 1: BrdU-positive cells in a coronal section (rat, male, Sprague–Dawley, 35–40 d old, 10 × objective) of the dentate gyrus.
Figure 2: Ki67-positive cells in a coronal section (rat, male, Sprague–Dawley, 35–40 d old, 10 × objective) of the dentate gyrus.
Figure 3: Examples of BrdU (left) and Ki67 (right) positive nuclei shown at high magnification (100 ×).
Figure 4: Doublecortin-positive cells in a coronal section (rat, male, Sprague–Dawley, 35–40 d old, 10 × objective).
Figure 5: Doublecortin-positive cells.

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Acknowledgements

We thank CIHR (Canada) for financial support, and present and past members of Wojtowicz' lab for their contributions.

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  1. Department of Physiology, University of Toronto, Toronto, M5S 1A8, Ontario, Canada

    J Martin Wojtowicz

  2. Program in Neuroscience and Mental Health, The Hospital for Sick Children, Toronto, M5G 1X8, Canada

    Nohjin Kee

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Wojtowicz, J., Kee, N. BrdU assay for neurogenesis in rodents. Nat Protoc 1, 1399–1405 (2006). https://doi.org/10.1038/nprot.2006.224

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