May 06, 2025
Immunostaining of Brain Sections (100μm and 40μm)
- Pia Rodriguez1
- 1Duke University
External link: https://doi.org/10.1083/jcb.202410130
Protocol Citation: Pia Rodriguez 2025. Immunostaining of Brain Sections (100μm and 40μm). protocols.io https://dx.doi.org/10.17504/protocols.io.261ge876og47/v1
Manuscript citation:
Salazar MPR, Kolanukuduru S, Ramirez V, Lyu B, Manigault G, Sejourne G, Sesaki H, Yu G, Eroglu C (2025) Mitochondrial fission controls astrocyte morphogenesis and organization in the cortex. The Journal of Cell Biology 224(10). doi: 10.1083/jcb.202410130
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: May 05, 2025
Last Modified: May 06, 2025
Protocol Integer ID: 217754
Keywords: immunostaining, brain section
Funders Acknowledgements:
Howard Hughes Medical Institute
Aligning Science Across Parkinson’s Initiative
Grant ID: ASAP-020607
BRAIN Initiative
Grant ID: U19-NS123719
Chan Zuckerberg Initiative, Neurodegeneration Challenge Network Collaborative
Grant ID: DAF2021-237435
Chan Zuckerberg Initiative, Neurodegeneration Challenge Network Collaborative
Grant ID: DAF2018-191999
HHMI Gilliam Fellowship
Grant ID: GT15076
Abstract
General Immunohistochemistry protocol
Troubleshooting
Prepare Solutions
Make TBST (0.2% Triton): 49ml 1x TBS + 1ml 10% TritonX (note: make solution fresh to begin your staining. Make sure that Triton bottle has not been open for more than 2-4 weeks)
Blocking and antibody solution: 10% goat serum in TBST
Note: All incubation and washing steps done with shaking.
Blocking and Primaries
Wash sections 3x 10 min in 1x TBS. 1ml of solution per well is sufficient for 4-6 sections per well in a 24 well plate.
Incubate in blocking solution (10% goat serum in TBST) for 1hr at room temperature.
Prepare primary antibody solution by diluting antibodies in 10% goat serum TBST solution. Vortex briefly to mix. Spin antibodies down at 5 mins, max speed. (This step is done to pellet any insoluble antibody aggregates that might cause background staining.)
Incubate in primary antibody solution at 4°C on shaker for at least 2 nights. Can incubate up to 3 nights.
Secondaries and Mounting
Incubate in secondary antibodies for 3hr at room temperature.
Wash in TBST 2 x 10 mins, 3rd rinse for 30 min.
Prepare secondary antibody solutions by diluting 1:200 in 10% goat serum TBST and spinning down as described with primary antibodies.
Wash in TBST 3x 10 mins.
One by one, transfer sections first to a container filled with 2/3 TBS, 1/3 ddH2O. Mount the sections (3 per slide) with the help of a clean brush on glass slides. Allow sections to dry for approximately 5-10 mins.
Use 1 drop of VectaShield mounting media (with DAPI) per section. Coverslip and then seal with nail polish.
Important! Wait at least 2hrs before imaging. This time is required for the sections to settle and achieve accurate z-stack imaging.