Jul 11, 2024
Immunostaining of Human Frontal Cortex Sections
- 1Duke University
Protocol Citation: Shiyi Wang 2024. Immunostaining of Human Frontal Cortex Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8mk5l5r/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: 103201
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Immunostaining of Human Frontal Cortex Sections
**Tissue Acquisition**
- Obtain 40 μm thick floating sections of human frontal cortex from Banner Sun Health Research Institute in Sun City, Arizona.
- Use sections from 4 control subjects and 3 LRRK2 G2019S mutation carrier subjects.
**Subject Information**
- Verify that control subjects had no history of dementia, neurological, or psychiatric disorders at the time of death (Refer to Supplemental Table 1).
- Ensure that informed and written consent was obtained from all donors.
**Preparation of Sections**
- Wash sections in 1x TBS containing 0.3% Triton X-100 (TBST) to remove any residual fixative and debris.
**Blocking**
- Block non-specific binding by incubating sections in 3% normal goat serum (NGS) diluted in TBST for blocking, typically for 1 hour at room temperature.
**Primary Antibody Incubation**
- Incubate sections overnight at 4°C with gentle shaking in primary antibodies diluted in blocking buffer: GFAP (chicken, 1:250; AB5541, Millipore Sigma), Phospho-ERM (Rabbit, 1:250; #3726, Cell Signaling)
**Washing**
- Wash sections thoroughly with TBST to remove unbound primary antibodies.
**Secondary Antibody Incubation**
- Incubate sections with Alexa Fluor conjugated secondary antibodies (diluted 1:200 in TBST) for 2-3 hours at room temperature in the dark.
**Final Washing**
- Wash sections again with TBST to remove unbound secondary antibodies.
**Mounting**
- Mount sections onto glass slides using a homemade mounting media composed of: 90% Glycerol, 20 mM Tris pH 8.0, 0.5% n-Propyl gallate
**Sealing**
- Seal coverslips with nail polish to prevent drying and movement during imaging.