Immunostaining of Human Frontal Cortex Sections
Shiyi Wang
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Abstract
Immunostaining of Human Frontal Cortex Sections
Steps
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.
