Jul 10, 2024
Human brain section staining
- 1Duke University
Document Citation: Shiyi Wang 2024. Human brain section staining. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4bb2gmk/v1
License: This is an open access document 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
Created: May 23, 2023
Last Modified: August 13, 2024
Document Integer ID: 82334
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
How to stain Human brain sections
1. Floating human frontal cortex sections of 40 μm thickness were obtained from Banner Sun Health Research Institute in Sun City, Arizona (4 control and 3 LRRK2 G2019S mutation carrier subjects).
- None of the control subjects had a history of dementia or neurological or psychiatric disorders at the time of death (See Supplemental Table 1). Informed and written consent was obtained from the donors.
2. For immunostaining, sections were washed in 1× TBS containing 0.3% Triton X-100 (TBST), blocked in 3% NGS diluted in TBST, and incubated in primary antibody 2-3 nights at 4°C with shaking.
3. Primary antibodies used were GFAP (chicken, 1:250; AB5541, Millipore Sigma) and phospho-ERM (Rabbit, 1:250; #3726, Cell Signaling).
4. Following primary incubation, sections were washed in TBST, incubated in Alexa Fluor conjugated secondary antibodies diluted 1:200 (Life Technologies) for 2-3 hours at room temperature, washed with TBST, and mounted onto glass slides using a homemade mounting media (90% Glycerol, 20 mM Tris pH 8.0, 0.5% n-Propyl gallate) and sealed with nail polish.