Apr 21, 2026
Astrocyte morphology assessment in astrocyte-neuron co-cultures
- 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. Astrocyte morphology assessment in astrocyte-neuron co-cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygxwobdv8j/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 21, 2026
Protocol Integer ID: 117980
Keywords: ASAPCRN, astrocyte morphology, sholl analysis, astrocyte-neuron co-culture, astrocyte morphology assessment in astrocyte, analyzing astrocyte morphology, astrocyte morphology assessment, astrocyte morphology in astrocyte, astrocyte
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
This protocol outlines the process for analyzing astrocyte morphology in astrocyte-neuron co-cultures using immunostaining and Sholl analysis.
Materials
- DPBS (GIBCO, Cat# 14190144)
- normal goat serum (NGS; Thermo Fisher, Cat# 01-6201)
- Triton X-100 Surfact-Amps Detergent Solution (Thermo Fisher, Cat# 28314)
- VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Cat# H-1200-10)
Antibodies
- chicken anti-GFP (1:1,000 dilution; Aves Labs, Cat# GFP-1020, RRID:AB_10000240)
- rabbit anti-GFAP (1:2,000 dilution; Agilent, Cat# Z0334, RRID:AB_10013382)
- Alexa Fluor 488 goat anti-chicken IgY(H+L) (1:1,000 dilution; Thermo Fisher, Cat# A-11039, RRID:AB_2534096)
- Alexa Fluor 594 goat anti-rabbit IgG(H+L) (1:1,000 dilution; Thermo Fisher, Cat# A-11037, RRID:AB_2534095)
Safety warnings
- Maintain sterility and avoid contamination during all steps.
- Follow institutional guidelines for the disposal of biological and chemical waste.
Fixation
7m
Fix astrocyte-neuron co-cultures on glass coverslips on Day in Vitro 12 (DIV 12) with warm 4% paraformaldehyde (PFA) for 00:07:00 at Room temperature .
7m
Wash coverslips 3 times with DPBS.
Blocking
30m
Block coverslips in a blocking buffer containing 50% normal goat serum (NGS) and 0.4% Triton X-100 for 00:30:00 at Room temperature .
30m
Primary antibody incubation
30m
Incubate samples Overnight at 4 °C in primary antibodies diluted in blocking buffer containing 10% NGS.
Note
Validate antibody specificity and optimize staining conditions for reproducibility.
30m
Remove primary antibody and wash coverslips three times with DPBS.
Secondary antibody incubation
2h
Incubate coverslips in Alexa Fluor conjugated secondary antibodies for 02:00:00 at Room temperature .
2h
Remove secondary antibody and wash coverslips three times with DPBS.
Mounting
7m
Mount coverslips onto glass slides using Vectashield mounting media containing DAPI.
Seal coverslips with nail polish.
Imaging
7m
Image coverslips using a Keyence BZ-X800 microscope at 40x magnification in red, green, and/or DAPI channels using a CCD camera.
Morphological analysis
7m
Analyze astrocyte morphological complexity using FIJI with the Sholl analysis plugin (https://github.com/Eroglu-Lab/In-Vitro-Sholl).
Note
Ensure analysis is performed on healthy astrocytes with strong expression of fluorescent markers and single, non-overlapping nuclei (DAPI stain).
Statistical analysis
7m
Conduct statistical analyses using custom code in R (https://github.com/Eroglu-Lab/In-Vitro-Sholl).
Use a mixed-effect model with Tukey post-test for Sholl analysis to evaluate differences between experimental conditions, treating variability per experiment as a random effect.
Quality control
7m
Verify the health of astrocyte-neuron co-cultures by ensuring the peak number of astrocyte intersections is ≥ 25 in the control condition.
Document the exact number of independent experiments and cells analyzed in the figure legend for each experiment.
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