Jul 10, 2024
Immunohistochemistry on mouse brain sections
- 1Duke University
Document Citation: Shiyi Wang 2024. Immunohistochemistry on mouse brain sections. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge3yywl47/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: July 10, 2024
Document Integer ID: 82330
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
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Abstract
Immunohistochemistry on mouse brain sections
1. Mice used for immunohistochemistry were anesthetized with 200 mg/kg Avertin and perfused with TBS/Heparin and 4% PFA.
2. Brains were collected and post-fixed in 4% PFA overnight, cryoprotected in 30% sucrose, frozen in a solution containing 2 parts 30% sucrose and 1-part O.C.T. (TissueTek), and stored at −80°C.
3. Floating coronal tissue sections of 30 μm, 40 μm or 100 μm thickness were collected and stored in a 1:1 mixture of TBS/glycerol at −20°C.
4. For immunostaining, sections were washed in 1× TBS containing 0.2% Triton X-100 (TBST), blocked in 10% NGS diluted in TBST, and incubated in primary antibody for 2-3 nights at 4°C with gentle shaking.
5. Primary antibodies used were anti-LRRK2 (Rabbit, 1:500; ab133474, Abcam), 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, Invitrogen), VGAT (Guinea pig, 1:1000; 131004, Synaptic Systems), and GEHPYRIN (Rabbit, 1:1000; #14304S, Cell Signaling).
6. Following the 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.
7. For DAPI staining, DAPI (1:50,000) was added to the secondary antibody solution for the final 10 minutes of incubation.
8. Images were acquired with an Olympus FV 3000 microscope.