Mar 30, 2026
IHF GAD67+ NeuN
- Aroa S Maroto1,
- Laura Perez-Cervera1,
- Silvia De Santis1
- 1Instituto de Neurociencias (Consejo Superior de Investigaciones Científicas - Universidad Miguel Hernández), San Juan de Alicante 03550, Spain
Protocol Citation: Aroa S Maroto, Laura Perez-Cervera, Silvia De Santis 2026. IHF GAD67+ NeuN. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbox8qlpk/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: January 19, 2026
Last Modified: March 30, 2026
Protocol Integer ID: 238909
Abstract
This is a double immunofluorescent staining method for GAD67 and NeuN in rat brain sections. This method is used to identify and quantify GABAergic neurons in relation to the total neuronal population. GAD67 (67kDa glutamate decarboxylase) is a specific marker for GABAergic inhibitory neurons, and NeuN is a pan-neuronal marker used to identify mature neurons.
Guidelines
This protocol is optimised for cryosections of 30 µm-thick rat brains that have been fixed in PFA and cryoprotected in 30% sucrose prior to embedding in OCT.
Materials
Materials
**EQUIPMENT**
- Multiwell 24 plate
- ThermoMixer
- 2ml microtubes
- Black tray
- Aluminum foil
- Tweezers
**CHEMICALS**
- Tri-sodium citrate Ref S4641
- PBS 1x
- Triton X-100 Ref: X100
- Tween20 Ref: P7949
- Goat Serum Ref: S26-M Sigma (GS)
- DAPI Ref: D9542
- VECTASHIELD‱ HardSet‱ Ref H-1400
**BUFFERS**
- Sodium Citrate Buffer (10mM Sodium Citrate + 0.05% Tween20 pH6)
- Wash buffer (Wb) PBS-Tx (PBS 1x + 0.5% Triton)
- Blocking Buffer (Wb + 5% GS)
- Antibody Diluent (Wb + 2.5% GS)
**ANTIBODIES**
- NeuN Merck MAB377 1/1000
- GAD67 Merck Ref. MAB5406
- Anti-Mouse 488 ThermoFisher Alexa Fluor‱ 488 A-11029 1/500
- Anti-Mouse 647 ThermoFisher Alexa Fluor‱ 647 A21236 1/500
**PROTECTIVE GEAR**
- Laboratory Coat
- Gloves
Troubleshooting
Safety warnings
DAPI is potentially mutagenic
Use laboratory coat and globes.
It is important that the tissue is not folded during the process, since this improves the antigen-antibody bond.
The order of the primary antibodies is extremely relevant: 1st GAD67, 2nd NeuN. Switching the order increases background noise and cross-talking between antibodies, making it difficult to differentiate and count cells. Furthermore, it is also important to select secondary antibodies with wavelengths that are as far as possible, again to facilitate the optimal differentiation between cells.
Ethics statement
All experimental procedures were approved by the Animal Care and Use Committee of the Instituto
de Neurociencias de Alicante (Spain) and were conducted in compliance with Spanish (Law 32/2007) and European regulations (EU Directive 86/609, EU Decree 2001-486, and EU Recommendation 2007/526/EC).
Before start
To succeed in this protocol, you need to have prior knowledge of the techniques involved in free flotation and immunohistochemistry, as well as knowing how to proceed. If you do not yet have the necessary experience, seek expert advice to learn the technical details.
Good luck!
Antigen Retrieval
1h
Select the slices to be performed and place one slice and citrate into 2 ml tubes. Place one slice in each tube and ensure they are completely submerged.
20m
Place the tubes in the thermoblock at 80ºC for 20 minutes. Shaking gently is not compulsory.
20m
Place around 3 slices/well on multi-well plate and wash with 500 ul wash buffer (Wb) 3 times x 5 min each. Kindly ensure that all the tissue in the well is thoroughly covered.
20m
Blocking and GAD67 antibody
3d 2h 25m
Add 500 μl/well of blocking Buffer 2 hours at RT with gentle shaking
2h
Remove the blocking buffer
10m
Prepare primary antibody dilution GAD67 1/500 in antibody diluent
15m
Add 500 μl/well of the primary antibody to each well and incubate at 4 °C with gentle shaking overweekend
3d
Secondary antibody
3h 20m
Remove GAD67 antibody
10m
Add 500 μl/well Wb and shaking incubation for 5 min and remove buffer (Repeat this step minimum 3 times)
25m
Prepare secondary antibody dilution Anti- Mouse 488 1/500
10m
Add 500 μl/well of the secondary antibody to each well and incubate at RT with gentle shaking for 2 hours.
2h
Remove secondary antibody
10m
Add 500 µl/well Wb and shaking incubation for 5 min and remove buffer (Repeat this step minimum 3 times).
25m
NeuN antibody
17h 20m
Prepare primary antibody dilution NeuN 1/1000 in antibody diluent
10m
Remove the Wb
10m
Add 500 μl/well of the primary antibody to each well and incubate at 4 °C with gentle shaking overweekend
17h
Secondary antibody
2h 45m
Remove primary antibody
10m
Add 500 µl/well Wb and shaking incubation for 5 min and remove buffer (Repeat this step minimum 3 times).
25m
Prepare secondary antibody dilution Anti- Mouse 647 1/500
10m
Add 500 μl/well of the secondary antibody to each well and incubate at RT with gentle shaking
2h
DAPI
1h 15m
Remove Secondary antibody
10m
Add 500 µl/well Wb and shaking incubation for 5 min and remove buffer (Repeat this step minimum 3 times)
25m
Add 500 µl/well of the DAPI to each well and incubate at RT with gentle shaking for 5 minutes
5m
Remove DAPI
10m
Add 500 µl/well Wb and shaking incubation for 5 min and remove buffer (Repeat this step minimum 3 times)
25m
Montage
25m
Bring the VECTASHIELD‱ HardSet‱ to room temperature
Mount the slices on the slides. Cover with approximately 50 µl of VECTASHIELD‱ HardSet‱ per slice
15m
Put the cover slide in a dark box and leave it to dry
10m
20
You have your slices ready for the microscope
Acknowledgements
The authors gratefully acknowledge the professional staff of the Imaging Facility at the Instituto de Neurociencias for their technical support and assistance in optimizing image acquisition and processing procedures.
The authors gratefully acknowledge the professional staff of the Animal Facility (SEA) of the Universidad Miguel Hernández (UMH) for their excellent animal care.