Feb 07, 2025
Version 2
Immunohistochemistry on mouse brain sections V.2
- 1Duke University
Protocol Citation: Shiyi Wang 2025. Immunohistochemistry on mouse brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8m4xl5r/v2Version created by Shiyi Wang
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: February 07, 2025
Protocol Integer ID: 119643
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Immunohistochemistry on mouse brain sections
**Anesthesia and Perfusion**
- Anesthetize mice with 200 mg/kg Avertin.
- Perform transcardial perfusion using TBS/Heparin followed by 4% paraformaldehyde (PFA).
**Brain Collection and Post-Fixation**
- Remove the brain and post-fix it in 4% PFA overnight at 4°C.
**Cryoprotection and Storage**
- Cryoprotect the brain in 30% sucrose solution until sunk.
- Embed the brain in a solution containing 2 parts 30% sucrose and 1 part O.C.T. (TissueTek).
- Store the brain blocks at −80°C until sectioning.
**Sectioning**
- Section the brain into 30 μm, 40 μm, or 100 μm thick coronal slices using a cryostat.
- Store the sections in a 1:1 mixture of TBS and glycerol at −20°C for further use.
**Preparation of Sections**
- Wash the brain sections in 1x TBS containing 0.2% Triton X-100 (TBST).
**Blocking**
- Block non-specific binding by incubating sections in 10% normal goat serum (NGS) diluted in TBST for 1 hour at room temperature.
**Primary Antibody Incubation**
- Incubate sections with primary antibodies diluted in blocking buffer (10% NGS in TBST) for 2-3 nights at 4°C with gentle shaking.
Primary antibodies used: - Anti-LRRK2 (Rabbit, 1:500; ab133474, Abcam), HA (Rat, 1:500; 11867423001, Roche), Phospho-ERM (Rabbit, 1:500; 3141, Cell Signaling), Sox9 (Rabbit, 1:500; AB5535, Millipore), GFAP (Rabbit, 1:500; Z0334, Agilent DAKO), VGluT1 (Guinea pig, 1:2000; 135304, Synaptic Systems), PSD95 (Rabbit, 1:300; 51-6900, Innovative Research), VGAT (Guinea pig, 1:1000; 131004, Synaptic Systems), Gephyrin (Rabbit, 1:1000; 147011, Synaptic Systems), S100β (Mouse IgG1, 1:200;S2532, Millipore Sigma), MAP2 (Mouse IgG1, 1:200; 13-1500, Thermo Fisher Scientific), and Iba1 (Chicken, 1:500; 234009, Synaptic Systems).
**Secondary Antibody Incubation**
- Wash sections with TBST.
- Incubate sections in Alexa Fluor conjugated secondary antibodies diluted 1:200 in TBST for 2-3 hours at room temperature.
**Mounting**
- Wash sections in TBST.
- Mount sections onto glass slides using a homemade mounting media (90% Glycerol, 20 mM Tris pH 8.0, 0.5% n-Propyl gallate).
- Seal the edges of the coverslip with nail polish.
**DAPI Staining**
- Add DAPI (1:50,000) to the secondary antibody solution for the final 10 minutes of incubation.
**Imaging**
- Acquire images using a fluorescence microscope (e.g., Olympus FV 3000).