May 06, 2025
Postnatal Astrocyte Labeling by Electroporation (PALE)
- Pia Rodriguez1
- 1Duke University
External link: https://doi.org/10.1083/jcb.202410130
Protocol Citation: Pia Rodriguez 2025. Postnatal Astrocyte Labeling by Electroporation (PALE). protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5op66g1b/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: May 06, 2025
Last Modified: May 06, 2025
Protocol Integer ID: 217753
Keywords: eroglu lab postnatal astrocyte labeling by electroporation, postnatal astrocyte labeling by electroporation, eroglu lab postnatal astrocyte labeling, postnatal astrocyte labeling, electroporation
Funders Acknowledgements:
Howard Hughes Medical Institute
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
Abstract
Eroglu Lab Postnatal Astrocyte Labeling by Electroporation (PALE) general protocol
Materials
Supplies needed:
- Electrodes
- Needles (pulled glass, as in electrophysiological experiments)
- DNA constructs (add small amount of Fast Green Dye for visualization)
- Mouth pipet
- Small ruler
- Marker
- P20 pipet and some tips
- Parafilm
- Transfer pipet
- Extra glove
- PBS in 15 ml tube
- Ice bucket
Troubleshooting
Set up
In the procedure room, wipe down your bench space with trifectant.
Place snuggle safe heating pad in microwave for 5:30 – 6:00 minutes.
Go get animals and bring them to procedure room.
Also get small spring scissors (for cutting toes) and small regular scissors (for cutting needle).
Take 2-3 pups from the cage and begin anesthetizing on ice.
Cut extra glove, place on top of ice and make a groove into the ice.
Lay mice on glove in the groove and fold glove over mice.
Pile ice on top of glove.
In ~5 minutes mice will be ready. Look for purple color and no movement.
While waiting for pups to be ready, continue with set-up.
Set up electroporator and check settings.
Mode: LV
Voltage: 100V
Pulse length: 50 ms
# pulses: 5
Interval: 950 ms
Polarity: Unipolar
Measure and mark needles using ruler. Every 3 mm is 1 µl.
Use scissors and make a diagonal cut to remove the bendy top of the needle.
Pipet ~8 µl of DNA onto parafilm.
Using mouth pipet, pull up ~3-4 µl into needle.
Electroporation
Once mouse is ready, place on bench top.
Using mouth pipet, inject 1 µl of DNA into lateral ventricle.
Use transfer pipet to wet the electrode with PBS.
Positive electrode has blue peg on it. Orient electrode so that the positive probe is on top of where DNA was injected (we are aiming for the visual cortex). The negative probe will be 180 degrees opposite, near the side of the jaw on the opposite side.
Cut toe to identify mouse if needed.
Record mouse ID and conditions.
Place mouse on snuggle safe to recover.
Mouse can go back to mom once it has regained pink color and is breathing and moving around.
Note: For CD1 mice, you can start anesthetizing the next 2-3 pups when you remove the first pups for electroporation. For other strains of mice, wait until your first 2 pups are ready to go back to mom before starting with more pups.