Jul 23, 2024
Immunohistochemistry of free floating slices
- 1Department of Pharmacology and Physiology and Department of Neurosciences, Faculty of Medicine, Université de Montréal, Montreal, QC, Canada. SNC and CIRCA Research Groups, Université de Montréal, Montréal, QC, Canada. Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815, USA.
Protocol Citation: louis-eric.trudeau Trudeau 2024. Immunohistochemistry of free floating slices. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzj6rrlx1/v1
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: June 20, 2024
Last Modified: July 24, 2024
Protocol Integer ID: 102402
Keywords: ASAPCRN
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Abstract
This protocol details the immunohistochemistry of free floating slices.
Steps
Steps
2w 0d 5h 40m
2w 0d 5h 40m
Note
Step 1 is for fresh tissue only, otherwise, start directly to step 2
Fix with paraformaldehyde (PFA) 4% Overnight at Room temperature .
- 4 g of PFA
- Dissolve in 100 mL de PBS (pH 7.3 ) previously heated to around 60 °C .
- Mix the solution until it completely clears up.
- Filter and store at 4 °C for a duration of 336:00:00 .
2w
Rinse with PBS 1X (3X 10 min) under agitation.
Rinse with PBS 1X for 00:10:00 under agitation (1/3).
10m
Rinse with PBS 1X for 00:10:00 under agitation (2/3).
10m
Rinse with PBS 1X for 00:10:00 under agitation (3/3).
10m
Permeabilize and block the non-specific primary antibodies bindings in a 24-well multiwell plate
by incubating under agitation for 01:00:00 with 0.5 mL to 1 mL of a BSA enriched ab solution (10 mL of ab solution for 1 g of BSA).
1h
All the subsequent steps are done using the following ab solution:
- 95 mL of PBS 7.3
- 20 mg of NaN3 (final 0.02 %)
- 300 µL of Triton X-100 (final 0.3 %)
- 500 mg of BSA (final 0.5 %)
- 5 mL of goat serum (final 5 %)
Dilute the primary antibodies in the ab solution and apply 0.5 mL per well (up to 10 slices)
Overnight at Room temperature under agitation.
1h
Rinse the slices with PBS (3X 10 min) under agitation.
Rinse the slices with PBS for 00:10:00 under agitation (1/3).
10m
Rinse the slices with PBS for 00:10:00 under agitation (2/3).
10m
Rinse the slices with PBS for 00:10:00 under agitation (3/3).
10m
Dilute the secondary antibodies in the ab solution and apply 0.5 mL per well (up to 10 slices) for 02:00:00 at Room temperature under agitation.
2h
Rinse the slices with PBS (3X 10 min) under agitation.
Rinse the slices with PBS for 00:10:00 under agitation (1/3).
10m
Rinse the slices with PBS for 00:10:00 under agitation (2/3).
10m
Rinse the slices with PBS for 00:10:00 under agitation (3/3).
10m
Mount the slices on charged (+) glass slides submerged in PBS.
Let the mounted slices dry (about 00:10:00 ).
Note
*Don’t wait too long otherwise salt deposits will start to appear and affect your staining.
10m
Seal the slides with 100 µL -120 µL /slide of Fluoromount G and a rectangular glass coverslip and seal the coverslip to the slide with nail polish.