Labeling Mitotically Active Cells in Cochlea

To label mitotically active cells, a single, subcutaneous injection of the thymidine analog 5-ethynyl-2′-deoxyuridine (EdU; 50 mg/kg) in DMSO was administered to DN-CBRb⁺/ROSA-CAG-rtTA⁺ and DN-CBRb⁺/ROSA-CAG-rtTA^- mice 4 h before tissue harvesting. EdU incorporation into DNA of whole-mount cochlea was detected using the Click-iT EdU Alexa 488 Fluor Imaging kit (Invitrogen) and double stained with DAPI following the manufacturer’s instructions and experimental procedures previously described (Kaiser et al., 2009 (link); Rocha-Sanchez et al., 2011 (link)). Samples were imaged using a Leica TCS SP8 MP confocal microscope.

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Tarang S., Doi S.M., Gurumurthy C.B., Harms D., Quadros R, & Rocha-Sanchez S.M. (2015). Generation of a Retinoblastoma (Rb)1-inducible dominant-negative (DN) mouse model. Frontiers in Cellular Neuroscience, 9, 52.

Publication 2015

TCS SP8 MP confocal microscope

Cells Cochlea Confocal microscope Dapi Deoxyuridine Dmso Mice Rosa Subcutaneous injection Thymidine

Corresponding Organization :

Other organizations : Creighton University, University of Nebraska Medical Center

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Variable analysis

independent variables

Administration of 5-ethynyl-2′-deoxyuridine (EdU; 50 mg/kg) in DMSO via a single, subcutaneous injection

dependent variables

EdU incorporation into DNA of whole-mount cochlea

control variables

Time between EdU administration and tissue harvesting (4 h)

controls

Positive control: DN-CBRb+/ROSA-CAG-rtTA+ mice
Negative control: DN-CBRb+/ROSA-CAG-rtTA- mice

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