Feb 05, 2026
Version 2
Immunohistochemical staining for GAD67 and TH in FFPE sections. V.2
- 1Brain and Mind Centre & Faculty of Medicine and Health School of Medical Sciences, The University of Sydney, Sydney, NSW 2050, Australia;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD, 20815
Protocol Citation: Anastasia Filimontseva, YuHong Fu, Glenda Halliday 2026. Immunohistochemical staining for GAD67 and TH in FFPE sections. . protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwnm57vmk/v2Version created by Anastasia Filimontseva
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: February 05, 2026
Last Modified: February 05, 2026
Protocol Integer ID: 242663
Keywords: ASAPCRN, gaba neurons in ffpe midbrain, immunohistochemical staining for gad67, gaba neuron, ffpe midbrain, immunohistochemical staining, dopamine, gad67, th in ffpe section, immunohistochemistry protocol, ffpe section
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-020505
National Health and Medical Research Council Investigator Grant
Grant ID: 1176607
Abstract
Immunohistochemistry protocol to identify dopamine and GABA neurons in FFPE midbrain human sections.
Materials
- FFPE tissue blocks
- Microscope slides
- D-Limonene (Hurst Scientific, Histo-5LCTN)
- Ethanol (100%, 95%, 70%)
- Distilled H2O
- Antigen retrieval solution (Invitrogen, 00-4956-58)
- Antigen retrieval cooker (Aptum Biologics Ltd, UK, Retriever 2100)
- Tris-buffered saline (TBS, Sigma-Aldrich, 94158-10TAB)
- Endogenous alkaline phosphatase (AP)
- Hydrogen peroxidase (HRP)
- BLOXALL blocking solution (SP-6000-100)
- Normal horse serum blocking solution (2% horse serum, 1% BSA in 0.01M TBS)
- Antibodies to tyrosine hydroxylase (TH, rabbit polyclonal IgG, AB152 Sigma-Aldrich, RRID:AB_390204)
- Antibodies to GAD67 (mouse monoclonal IgG3, sc-28376 Santa-Cruz Biotechnology, RRID:AB_627650)
- ImmPRESS™-alkaline phosphatase (AP) horse anti-mouse IgG polymer detection kit (MP-5402 Vector Laboratories, RRID:AB_2336535)
- ImmPRESS™-horseradish peroxidase (HRP) horse anti-rabbit IgG polymer detection kit (MP-7401 Vector Laboratories, RRID:AB_2335333)
- SignalStain® Vibrant Red AP Substrate Kit (76713 Cell Signaling)
- ImmPACT® DAB EqV HRP Substrate Kit (SK-4103 Vector Laboratories)
- Cresyl violet (HB5608 Hello Bio)
- Xylene
- Eukitt® quick-hardening mounting medium (03989 Sigma-Aldrich)
Paraffin removal and section rehydration
After standard human brain tissue processing (10.17504/protocols.io.8epv55dxdv1b/v1), sections were deparaffinized by incubation in 60°C oven for 1 hour.
Sections were submerged in D-Limonene (Hurst Scientific, Histo-5LCTN) for 2 x 7 minutes.
Sections were rehydrated in decreasing ethanol concentrations (100% ethanol for 2 x 3 minutes, 95% ethanol for 3 minutes, 70% ethanol for 3 minutes).
Sections were placed in distilled H2O for 3 minutes.
Antigen Retrieval
Sections were incubated in 1x antigen retrieval solution (HIAR) (Invitrogen, 00-4956-58) in a programmable antigen retrieval cooker (Aptum Biologics Ltd, UK, Retriever 2100).
After reaching peak temperature at ~121°C, sections were gradually cooled for 2 hours.
Blocking
Sections were washed in tris-buffered saline (TBS, Sigma-Aldrich, 94158-10TAB) for 5 minutes.
Sections were washed with TBS with Tween-20 (TBST) (Sigma, P1379) for 5 minutes.
After removing liquid from the slides hydrophobic circles were carefully drawn with A-PAP pen (Daido Sangyo) without touching the tissue sections.
Sections were blocked with BLOXALL blocking solution (SP-6000-100) for 30 minutes to quench endogenous alkaline phosphatase (AP) and hydrogen peroxidase (HRP).
Sections were blocked again with in-house made normal horse serum blocking solution (2% horse serum, 1% BSA in 0.01M TBS) for 1 hour at room temperature.
Primary antibody incubation
A primary antibody cocktail of tyrosine hydroxylase (TH, rabbit polyclonal IgG, AB152 Sigma-Aldrich, RRID:AB_390204, diluted 1:200) and GAD67 (mouse monoclonal IgG3, sc-28376 Santa-Cruz Biotechnology, RRID:AB_627650, diluted 1:50) were made in the same normal horse serum blocking solution.
Sections were incubated with this primary cocktail for 4 nights at 4°C.
Washing
Sections were washed in TBST 3x 10 minutes.
Secondary antibody incubation
Secondary antibody cocktail was made by adding equal volumes of ImmPRESS™-alkaline phosphatase (AP) horse anti-mouse IgG polymer detection kit (MP-5402 Vector Laboratories, RRID:AB_2336535) and ImmPRESS™-horseradish peroxidase (HRP) horse anti-rabbit IgG polymer detection kit (MP-7401 Vector Laboratories, RRID:AB_2335333).
Sections were incubated in the secondary antibody solution for 90 minutes at room temperature.
Secondary AP antibody development
Sections were washed 3x 5mins in TBST.
Sections were placed in distilled H2O for 5 minutes and the SignalStain® Vibrant Red AP Substrate Kit (76713 Cell Signaling) solution was prepared during this time for AP secondary visualisation.
2 drops of reagent A and 2 drops of reagent B were added in 5ml of diluent of the SignalStain® Vibrant Red AP Substrate Kit (76713 Cell Signaling).
The SignalStain® Vibrant Red AP solution was added to sections for approximately 15 minutes.
Secondary HRP antibody development
Stained sections were placed in distilled H2O to stop the reaction.
Sections were placed under running tap water for a few minutes and placed back into distilled H2O.
The ImmPACT® DAB EqV HRP Substrate Kit (SK-4103 Vector Laboratories) was prepared during this time.
Equal volumes of ImmPACT® DAB EqV reagent 1 and ImmPACT® DAB EqV reagent 2 were added and mixed.
The ImmPACT® DAB EqV solution was added to sections for approximately 1-2 minutes.
Sections were placed in distilled H2O to stop the reaction. Sections were then placed under running water, and after a few minutes returned to distilled H2O.
Cresyl violet counterstaining
Sections were placed in 0.5% cresyl violet (HB5608 Hello Bio) for 10 minutes.
Dehydration
Sections were dehydrated by incubation in increasing ethanol concentrations.
Sections were submerged for 3 minutes in 70%, 3 minutes in 90% and 2x 3 minutes in 100% ethanol.
Clearing and mounting
Dehydrated sections were cleared by submerging in xylene 2x 3 minutes.
Sections were mounted with Eukitt® quick-hardening mounting medium (03989 Sigma-Aldrich).