Jul 11, 2024
Immunofluorescence Staining and Analysis of Astrocyte-Neuron Co-Cultures
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Immunofluorescence Staining and Analysis of Astrocyte-Neuron Co-Cultures. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v923j4l3e/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: 103194
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Immunofluorescence Staining and Analysis of Astrocyte-Neuron Co-Cultures
**Fixation**
- Fix astrocyte-neuron co-cultures on glass coverslips on Day in Vitro 12 (DIV 12) with warm 4% paraformaldehyde (PFA) for 7 minutes.
- Wash coverslips 3 times with phosphate-buffered saline (PBS).
**Blocking**
- Block coverslips in a blocking buffer containing 50% normal goat serum (NGS) and 0.4% Triton X-100 for 30 minutes at room temperature.
- Wash coverslips with PBS.
**Primary Antibody Incubation**
- Incubate samples overnight at 4°C in primary antibodies diluted in blocking buffer containing 10% NGS.
- Incubate coverslips in Alexa Fluor conjugated secondary antibodies (Life Technologies) for 2 hours at room temperature.
- Wash coverslips again with PBS.
**Mounting**
- Mount coverslips onto glass slides (VWR Scientific) using Vectashield mounting media containing DAPI (Vector Labs).
- Seal coverslips with nail polish.
**Imaging**
- Image coverslips using an AxioImager M1 fluorescence microscope (Zeiss) at 40x magnification in red, green, and/or DAPI channels using a CCD camera.
**Morphological Analysis**
- Analyze astrocyte morphological complexity using FIJI with the Sholl analysis plugin (https://github.com/Eroglu-Lab/In-Vitro-Sholl).
- Ensure analysis is performed on healthy astrocytes with strong expression of fluorescent markers and single, non-overlapping nuclei (DAPI stain).
**Statistical Analysis**
- 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**
- 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.
Notes:
- Maintain sterility and avoid contamination during all steps.
- Ensure imaging settings are consistent across experimental conditions for accurate comparison.
- Validate antibody specificity and optimize staining conditions for reproducibility.