May 01, 2025
EdU Click-iT Administration
- Pia Rodriguez1
- 1Duke University
External link: https://doi.org/10.1083/jcb.202410130
Protocol Citation: Pia Rodriguez 2025. EdU Click-iT Administration. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5m5bdl1b/v1
Manuscript citation:
Salazar MPR, Kolanukuduru S, Ramirez V, Lyu B, Manigault G, Sejourne G, Sesaki H, Yu G, Eroglu C (2025) Mitochondrial fission controls astrocyte morphogenesis and organization in the cortex. The Journal of Cell Biology 224(10). doi: 10.1083/jcb.202410130
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 30, 2025
Last Modified: May 01, 2025
Protocol Integer ID: 203629
Keywords: EdU, imaging kit, thermo fisher click, imaging, it administration this protocol, c10640, fluorophore, protocol
Funders Acknowledgements:
Paul and Daisy Soros Fellowship
HHMI Gilliam Fellowship
Grant ID: GT15076
Chan Zuckerberg Initiative, Neurodegeneration Challenge Network Collaborative
Grant ID: DAF2018-191999
Chan Zuckerberg Initiative, Neurodegeneration Challenge Network Collaborative
Grant ID: DAF2021-237435
BRAIN Initiative
Grant ID: U19-NS123719
Aligning Science Across Parkinson’s initiative
Grant ID: ASAP-020607
Howard Hughes Medical Insitute
Abstract
This protocol uses the Thermo Fisher Click-iT EdU Imaging Kit (I used the 647 fluorophore, C10640, https://www.thermofisher.com/order/catalog/product/C10640).
Materials
Thermo Fisher Click-iT EdU Imaging Kit
Troubleshooting
Prepare EdU Stock Solution
Add 2 ml of DPBS to 5 mg EdU stock vial (component A).
Aliquot into 100 µl aliquots and store at -80°C.
This makes a 10 µM solution of EdU (2.5 µg/µl).
EdU Injection into Pups - IP Injection
Must inject 10-15 µg of EdU/gram of animal weight, so 4-6 µl of working 10 µM stock per gram of animal weight.
Thaw out appropriate number of aliquots depending on weight and number of pups/mice you wish to inject. Combine into one vial and take to the mouse house. For CD1/bl6 pups, P3 pups weigh ~3 g for example.
Pull all volume into syringe to inject pups as you weigh them. I inject 6 µl/g with a 2 ml syringe (this syringe uses ~70 µl of void volume, so add that to your total volume).
Weigh one pup at a time and inject IP appropriate volume (I round up for injection, i.e., 3 g pup would get 18 µl of 10 µM stock, so 20 µl injection).
Tissue Collection and Staining
Collect tissue as normal, section, block with NGS.
After blocking, follow Click-iT EdU Thermo Fisher staining protocol (Starting with “Detect EdU section, step 4.1).
EdU staining takes ~1 hr.
After EdU staining, can continue with regular primary antibody incubation/staining protocol as needed.