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J Neurocytol 18:311–318Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, Kashiwagi S, Fukami K, Miyata T, Miyoshi H et al (2008) Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. EdU labeling of proliferating cells in whole mount and sectioned embryos.
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E(�P��M{ ��������2 After use, store any remaining solutions at 2–6˚C. Growth medium, cell density, cell type variations, and other factors may influence labeling. Growth medium, cell density, cell type variations, and other factors may influence labeling. Inject 0.2 ml for each mouse every 24 hrs, or 0.1 ml every 12 hrs. 0000024589 00000 n
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This is in contrast to BrdU assays that require DNA denaturation (using acid, heat, or digestion with DNase) to expose the BrdU so that it may be detected with an anti-BrdU antibody.
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We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (“click” chemistry). However, we have not yet been able to establish a protocol that enables EdU to co-label with Phalloidin, a very popular marker for labeling F-actin in hair cells and supporting cells of the cochlea. 2 EdU labelling seems to cause DNA damage and activates the DNA damage checkpoint, making labelling experiments over more than one cell cycle problematic.
Subscription will auto renew annually.Over 10 million scientific documents at your fingertips Labeling Cells with EdU. Immunostaining. When stored as directed, this kit is stable for up to 1 year after receipt. Grow an overnight of MG1693 (thy-, from E. coli stock center)
J Neurosci 25:6533–6538Harris L, Zalucki O, Gobius I, McDonald H, Osinki J, Harvey TJ, Essebier A, Vidovic D, Gladwyn-Ng I, Burne TH et al (2016) Transcriptional regulation of intermediate progenitor cell generation during hippocampal development.
However the method by a practiced technician takes 2 hours and requires the harsh treatment of cells with 2M hydrochloric acid. Standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® detection reagent to gain access to the DNA. p���]�������?�O��ad�W�*,g��?�?���=�q8l���{^���vң3>{dl�
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���!2=�i�_���I�^XV��b>wŋF�Z������r>\�'�p��EP��*�Q@�}��n��h��E�Z��Jc���9r>u0O�mY���O���
����E/8��b�嗧g"{)��ޜ>}:��իG"���h��i"����iL�j;P��@�1��;ڗP��E"ؓ��,��I����pc!�h%��G�|�Fx�ZqrQ�d�f���ܺ�'���,��.� This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol. First, we determined the concentration of EdU needed to label whole mount chick embryos. Labeling protocol 1. 0000002959 00000 n
After washing in 2X SSC for 10 minutes, the standard immuno-labelling protocols were applied.
Technical Hints This kit is sold based on number of tests. For shorter incubations, higher concentrations may be required. 0000424780 00000 n