Oct 20, 2022

Public workspaceImmunofluorescent Labelling of Post-Mortem Rodent Brain Tissue V.1

DOI
dx.doi.org/10.17504/protocols.io.j8nlkkq31l5r/v1
  • 1Department of Physiology, Anatomy and Genetics, Oxford University, Oxford UK;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD
Cláudia C. Mendes
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DOI: dx.doi.org/10.17504/protocols.io.j8nlkkq31l5r/v1
Protocol Citation: Jeff Stedehouder, Shinil Raina, Stephanie J Cragg 2022. Immunofluorescent Labelling of Post-Mortem Rodent Brain Tissue. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkkq31l5r/v1
Manuscript citation:
https://pubmed.ncbi.nlm.nih.gov/28922832/
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 19, 2022
Last Modified: October 20, 2022
Protocol Integer ID: 67073
Keywords: Histology, immunofluorescence, postmortem, immunohistochemistry
Funders Acknowledgement:
Medical Research Council (MRC)
Grant ID: MR/V013599/1
Disclaimer
Special thanks to Elize Haasdijk and Erika Sabel-Goedknegt for help with very early versions of this protocol
Abstract
This protocol is to label one or several targets using a two-step immunofluorescent approach in PFA perfusion-fixed, 50-µm thick, post-mortem rodent brain tissue.
Note
Steps 24-26 of this protocol (DAPI labelling) are optional. DAPI is a stain to label nuclei of all cells (neurons, glia) in the tissue. Strongly recommended for independent identification of individual cells and for topographical orientation.

Guidelines
Every antibody and target has its own optimal binding conditions. Several portions of this protocol could be optimized for specific labelling targets.
Materials
Equipment:

Reagents:

Washes
Washes
45m
45m
Place 50 µm thick brain slices in a jar (or well). The jar or well should be able to hold ~2-5 ml Phosphate Buffered Saline (PBS) for proper washing.
Note
The volume of solutions specified in this protocol are recommended for a maximum of 12 slices. If using more slices than 12, it is suggested to increase the preincubation volume to 2 ml and the incubation for primary and secondary antibodies to 1.5 ml.



Add a volume (~2 ml) of PBS in each jar (or well).
Note
Unless specified, all these steps should be done at room temperature (20-22 °C)


Place the jar or well on an orbital shaker for ~10-15 mins.
Note
Shaker speed should be sufficient for the slices to move but not damage from the shaking. Be careful not to damage the slices or leave them dry without any buffer for too long.

After ~10-15 mins, remove the majority of PBS and add fresh PBS.
Repeat wash (steps 3 and 4) two more times for 10-15 mins each.
Preincubation
Preincubation
1h
1h
Prepare preincubation solution containing PBS with 10% Normal Bovine Serum (NBS) and 0.5% Triton-X (total 1.5 ml per well/jar) during the last wash.
Remove the PBS from the last wash and add the preincubation solution.
Note
Make sure all slices are in the solution and freely moving (e.g. not stuck to side of well/jar).

Leave to preincubate at room temperature for 1 h on an orbital shaker.
Primary Antibody
Primary Antibody
2d
2d
Prepare 2% Normal Bovine Serum (NBS) and 0.4% Triton-X (total 1 ml per well/jar) during the preincubation period.
Add the Primary Antibody to the above mix in the desired concentration. For instance, the S100β and ChAT antibodies were used at 1:1000 and 1:100, respectively.
Once done, remove all the preincubation solution and add the Primary Antibody Solution.
Note
Make sure all slices are in the solution and freely moving (e.g. not stuck to side of well/jar).

Leave to incubate for ~48-72 h at 4º C on an orbital shaker.
Washes
Washes
45m
45m
Remove all the Primary Antibody Solution and add fresh PBS.
Place the jar or well on an orbital shaker for ~10-15 mins.
Note
Shaker speed should be sufficient for the slices to move but not damage from the shaking. Be careful not to damage the slices or leave them dry without any buffer for too long.

After ~10-15 mins. remove the majority of PBS and add fresh PBS.
Repeat wash (steps 14 and 15) two more times for 10-15 mins each.
Secondary Antibody
Secondary Antibody
2h
2h
Prepare PBS with 2% Normal Bovine Serum (NBS) and 0.4% Triton-X (total 1 ml per well/jar) during the last wash.
Add the Secondary Antibody to the above mix in the desired concentration (commonly 1:300). For instance, Donkey anti-Guinea pig Alexa Fluor 488 and Donkey anti-Goat Dylight 594 were used at 1:300.
Once done, remove all the PBS from the last wash and add the Secondary Antibody Solution.
Note
Make sure all slices are in the solution and freely moving (e.g. not stuck to side of well/jar).

Leave to incubate for ~2-5 h at room temperature on an orbital shaker.
Washes
Washes
20m
20m
Remove all the Secondary Antibody Solution and add fresh Phosphate buffer (PB).
Place the jar or well on an orbital shaker for ~10 mins.
Note
Shaker speed should be sufficient for the slices to move but not damage from the shaking. Be careful not to damage the slices or leave them dry without any buffer for too long.

After ~10 minutes, remove the majority of PB and add fresh PB.
Repeat wash (steps 22 and 23) one more time for ~10 mins.
DAPI
DAPI
15m
15m
Prepare DAPI solution by mixing 1 µl DAPI Readymade solution in 1 ml PB (1:1000).
Optional
Remove the PB from the last wash and add the DAPI Solution.
Leave on the orbital shaker for ~15 mins.
Note
Shaker speed should be sufficient for the slices to move but not damage from the shaking. Be careful not to damage the slices or leave them dry without any buffer for too long.

Washes
Washes
20m
20m
Remove all the DAPI Solution and add fresh PB.
Place the jar or well on an orbital shaker for ~10 mins.
Note
Shaker speed should be sufficient for the slices to move but not damage from the shaking. Be careful not to damage the slices or leave them dry without any buffer for too long.

After ~10 minutes, remove the majority of PB and add fresh PB.
Repeat wash (steps 29 and 30) one more time for ~10 mins.
Mounting
Mounting
Mount the individual slices on a glass microscope slide.
Apply a small drop of Vectashield on the glass coverslip, and carefully place the coverslip on the glass slides containing the brain slices.
Add nail polish to the edges to seal the Vectashield shut and leave to dry before long-term storage in the fridge.
Note
Proceed with imaging within days after completing the protocol. Under standard conditions - assuming the protocol was properly executed - the samples should remain in imaging conditions for months/up to a year, although the signal will fade over time.