Apr 15, 2026
Postnatal astrocyte labeling by electroporation (PALE)
- 1Duke University;
- 2KU Leuven;
- 3Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Shiyi Wang, Sarah van Veen 2026. Postnatal astrocyte labeling by electroporation (PALE). protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj9z4xlk5/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: January 09, 2025
Last Modified: April 15, 2026
Protocol Integer ID: 117987
Keywords: ASAPCRN, PALE, Postnatal Astrocyte Labeling by Electroporation, postnatal astrocyte labeling by electroporation, postnatal astrocyte labeling, investigating astrocyte biology, astrocyte biology, electroporation procedure, electroporation, neurodevelopment, role in neurodevelopment
Funders Acknowledgements:
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only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind.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
Here, we provide a step-by-step guide to perform Postnatal Astrocyte Labeling by Electroporation (PALE), detailing the preparation, electroporation procedure, and post-electroporation analysis. This technique represents a powerful tool for investigating astrocyte biology and their role in neurodevelopment and disease.
Materials
- CD1 mice (Charles River, RRID:IMSR_CRL:022)
- pGLAST-PBase
- pPB-shRNA-mCherryCAAX
- FastGreen Dye
- mouth pipette (Drummond Scientific)
- Tissue-Tek Optimal Cutting Temperature (OCT) solution (Cat# 4583; Electron Microscopy Sciences)
- rabbit anti-RFP primary antibody (Rockland, Cat# 600-401-379, RRID:AB_2209751)
- Alexa Fluor 594 goat anti-rabbit IgG(H+L) secondary antibodies (Thermo Fisher, Cat# A-11037, RRID:AB_2534095)
- DAPI (Invitrogen, Cat# D1306)
- Fluorescence microscope (e.g. Olympus FV 3000)
- Imaris Bitplane 9.9
Safety warnings
- Ensure all procedures are performed in compliance with institutional guidelines for animal care and use.
- Follow institutional guidelines for the disposal of biological and chemical waste.
Animal preparation
Anesthetize late P0/early P1 wild-type CD1 mice by hypothermia.
Plasmid preparation
Prepare plasmid DNA mixed with Fast Green Dye for visualization.
Note
For shRNA knockdown experiments: Prepare 1 μl of DNA containing 1 μg of pGLAST-PBase and 1 μg of pPB-shRNA-mCherryCAAX for injection.
Injection procedure
Inject 1 μl of plasmid DNA mixture into the lateral ventricle of one hemisphere using a pulled glass pipette (Drummond).
Note
Maintain sterile conditions during plasmid preparation and injection procedures.
Electroporation
Orient electrodes with the positive terminal above the visual cortex and the negative terminal below the chin of the pups.
Apply 5 discrete 50 ms pulses of 100 V spaced 950 ms apart.
Note
Optimize electroporation parameters for consistent and reproducible results.
Cut toe to identify mouse if needed.
Record mouse ID and conditions.
Recovery
Recover pups on a heating pad. Mouse can go back to home cage once it has regained pink color and is breathing and moving around.
Monitor pups until collection at P21.
Anesthesia and perfusion
Anesthetize mice with 200 mg/kg Avertin.
Perform transcardial perfusion using TBS/Heparin followed by 4% paraformaldehyde (PFA).
Brain collection and post-fixation
1h
Remove the brain and post-fix it in 4% PFA Overnight at 4°C.
1h
Cryoprotection and storage
Cryoprotect the brain in 30% sucrose solution until sunk.
Embed the brain in a solution containing 2 parts 30% sucrose and 1 part O.C.T. (TissueTek).
Store the brain blocks at −80°C until sectioning.
Sectioning
Section the brain into 100 μm thick coronal slices using a cryostat.
Store the sections in a 1:1 mixture of TBS and glycerol at −20°C for further use.
Permeabilization of sections
Wash the brain sections in 1x TBS containing 0.2% Triton X-100 (TBST).
Note
- Use brain sections containing the visual cortex.
- Examine brain sections for the presence of electroporated cells before subsequent staining procedures.
Blocking
1h
Block non-specific binding by incubating sections in 10% normal goat serum (NGS) diluted in TBST for 01:00:00 at Room temperature .
1h
Antibody incubation
3h
Incubate sections with rabbit anti-RFP primary antibody (1:1,000) diluted in blocking buffer (10% NGS in TBST) for 3 nights at 4 °C with gentle shaking.
Wash sections with TBST.
Incubate sections in Alexa Fluor 594 goat anti-rabbit IgG(H+L) secondary antibodies diluted 1:200 in TBST, for 03:00:00 at Room temperature .
3h
Wash sections with TBST (3x 10 min).
Note
For nuclear staining, add DAPI (1:50,000) to the second wash.
Mounting
2h
One by one, transfer sections first to a container filled with 2/3 TBS, 1/3 ddH2O. Mount the sections (3 per slide) with the help of a clean brush on glass slides. Allow sections to dry for approximately 5-10 min.
Mount sections onto glass slides using a homemade mounting media (90% Glycerol, 20 mM Tris pH 8.0, 0.5% n-Propyl gallate).
Seal the edges of the coverslip with nail polish.
Important! Wait at least 02:00:00 before imaging. This time is required for the sections to settle and achieve accurate z-stack imaging.
2h
Imaging
3h
Acquire images using a fluorescence microscope (e.g., Olympus FV 3000) with a 60x objective, 2x zoom to acquire high-magnification and resolution (0.5 μm step size) images of individual astrocytes.
Note
Criteria for inclusion: Capture the entire astrocyte within a single brain section.
Analysis
3h
Surface reconstructions
Analyze imaged astrocytes using Imaris Bitplane software.
Generate surface reconstructions of the astrocytes.
Territory volume measurement
Use Imaris Xtensions “Visualize Surface Spots” and “Convex Hull” to create additional surface renders representing the territory of each astrocyte.
Record the volume of each astrocyte territory.
Statistical testing
Analyze astrocyte territory sizes from biological replicates across experimental conditions.
Use a nested t test or nested two-way ANOVA followed by the Bonferroni posthoc test for statistical comparisons.
Protocol references
Christabel Xin Tan, Dhanesh Sivadasan Bindu, Evelyn J. Hardin, Kristina Sakers, Ryan Baumert, Juan J. Ramirez, Justin T. Savage, Cagla Eroglu; δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte–neuron cadherin interactions. J Cell Biol 6 November 2023; 222 (11): e202303138. doi: https://doi.org/10.1083/jcb.202303138
Katherine T. Baldwin, Christabel X. Tan, Samuel T. Strader, Changyu Jiang, Justin T. Savage, Xabier Elorza-Vidal, Ximena Contreras, Thomas Rülicke, Simon Hippenmeyer, Raúl Estévez, Ru-Rong Ji, Cagla Eroglu,
HepaCAM controls astrocyte self-organization and coupling, Neuron, Volume 109, Issue 15, 2021, Pages 2427-2442.e10, ISSN 0896-6273, https://doi.org/10.1016/j.neuron.2021.05.025.
Stogsdill, J., Ramirez, J., Liu, D. et al. Astrocytic neuroligins control astrocyte morphogenesis and synaptogenesis. Nature 551, 192–197 (2017). https://doi.org/10.1038/nature24638