To permanently label newly generated cortical neurons, pregnant dams at E16.5 were single-dosed with 5–ethynyl–2′–deoxyuridine (EdU, Cayman Chem) in 1X PBS at 30mg/kg body weight via intraperitoneal injection. Pups were perfused at P14 and sectioned as described above. EdU detection was conducted upon the completion of immunofluorescence labeling. After washing in PBS for 10 minutes, brains sections were incubated with Alexa 647-conjugated Azide (Biotium) at 1.6μM, in solution containing 0.1M Tris-HCl (pH 8.8), 0.43 X PBS, 4 mM CuSO4, and 0.1M Ascorbate, for 20 minutes. Brains sections were washed with PBS/T three times at 20 minutes each, stained with DAPI and mounted for confocal imaging.
Alexa 647 conjugated azide
Manufactured by Biotium
Alexa 647-conjugated Azide is a fluorescent labeling reagent that can be used to detect and visualize azide-labeled biomolecules such as proteins, nucleic acids, and lipids. It consists of an azide group covalently linked to the Alexa Fluor 647 fluorescent dye, which emits light in the far-red region of the spectrum.
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2 protocols using alexa 647 conjugated azide
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Permanent Labeling of Cortical Neurons
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Permanent Labeling of Cortical Neurons
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To permanently label newly generated cortical neurons, pregnant dams at E16.5 were single-dosed with 5–ethynyl–2′–deoxyuridine (EdU, Cayman Chem) in 1X PBS at 30mg/kg body weight via intraperitoneal injection. Pups were perfused at P14 and sectioned as described above. EdU detection was conducted upon the completion of immunofluorescence labeling. After washing in PBS for 10 minutes, brains sections were incubated with Alexa 647-conjugated Azide (Biotium) at 1.6μM, in solution containing 0.1M Tris-HCl (pH 8.8), 0.43 X PBS, 4 mM CuSO4, and 0.1M Ascorbate, for 20 minutes. Brains sections were washed with PBS/T three times at 20 minutes each, stained with DAPI and mounted for confocal imaging.
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