Jul 11, 2024
Postnatal astrocyte labeling by electroporation (PALE)
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Postnatal astrocyte labeling by electroporation (PALE). protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyweprvx9/v1
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: July 11, 2024
Last Modified: July 11, 2024
Protocol Integer ID: 103195
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Postnatal astrocyte labeling by electroporation (PALE)
**Animal Preparation**
- Sedate late P0/early P1 mice by hypothermia until anesthetized.
**Plasmid Preparation**
- Prepare plasmid DNA mixed with Fast Green Dye for visualization.
**Injection Procedure**
- Inject 1 μl of plasmid DNA mixture into the lateral ventricle of one hemisphere using a pulled glass pipette (Drummond).
**shRNA Knockdown Experiments in Wild-Type CD1 Mice**
- Prepare 1 μl of DNA containing 1 μg of pGLAST-PBase and 1 μg of pPB-shRNA-mCherryCAAX for injection.
**Astrocyte Labeling in WT and LRRK2 G2019Ski/ki Mice**
- Prepare 1 μl of DNA containing 1 μg of pGLAST-PBase and 1 μg of pPB-mCherry-CAAX for injection per mouse.
**PALE-Mediated Overexpression of Phospho-mimetic Ezrin in shRNA Knockdown Experiments**
- Prepare 1 μl of DNA mixture containing 0.5 μg pGLAST-PBase, 0.5 μg pPB-shRNA-mCherryCAAX, and 1 μg pZac2.1-GfaABC1D-Ezrin T567D-BioID2-HA.
**Phospho-Dead Ezrin Overexpression in WT and LRRK2 G2019Ski/ki Mice**
- Prepare 1 μl of DNA mixture containing 0.5 μg pGLAST-PBase, 0.5 μg pZac2.1-gfaABC1D-mCherry-CAAX, and 1 μg pZac2.1-GfaABC1D-Ezrin T567A-BioID2-HA.
**Electrode Placement and Electroporation**
- Orient electrodes with the positive terminal above the frontal cortex and the negative terminal below the chin of the pups.
- Apply 5 discrete 50 ms pulses of 100 V spaced 950 ms apart.
**Recovery**
- Recover pups on a heating pad and return them to their home cage.
- Monitor pups until collection at P21.
**Animal Monitoring and Sample Collection**
- Monitor all animals for health status until collection at P21.
**Brain Section Examination**
- Examine brain sections for the presence of electroporated cells before subsequent staining procedures.
Notes:
- Ensure all procedures are performed in compliance with institutional guidelines for animal care and use.
- Maintain sterile conditions during plasmid preparation and injection procedures.
- Optimize electroporation parameters for consistent and reproducible results.