Apr 15, 2026

IHC staining (DAB) of Iba1 and Gfap

 Forked from IHC staining (DAB) of Iba1 and GFAP
DOI
https://dx.doi.org/10.17504/protocols.io.q26g7oj83vwz/v1
  • 1KU Leuven;
  • 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Sarah van Veen
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DOI: https://dx.doi.org/10.17504/protocols.io.q26g7oj83vwz/v1
Protocol Citation: Hanne Dhondt, Joris Van Asselberghs, Sarah van Veen 2026. IHC staining (DAB) of Iba1 and Gfap. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7oj83vwz/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: April 07, 2026
Last Modified: April 15, 2026
Protocol  Integer ID: 314579
Keywords: ihc staining, iba1, gfap this protocol detail, dab
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-020607
Abstract
This protocol details the IHC staining (DAB) of Iba1 and Gfap.
Attachments
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Protocol_DAB_general...
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Guidelines
General note:

Make sure that at each incubation step the tissue is flat and completely in touch with the liquid!!!

4 sections per animal



Materials
Materials
  • 24 well plates
  • oven (80 °C )
  • Ice
  • Wobbler
  • Falcon tubes (to prepare blocking and antibody dilutions)
  • Covering sleeves
  • Aluminium foil
  • 0.22 µm filter (Carl Roth; SE2M035I07)
  • 10 ml syringe
  • Plastic bag
  • Petri dish
  • Gelatine coated microscope slides
  • Cover slips #1.5 (Epredia; BB02400500SC13MNZ0)

Reagents
  • Citrate buffer 1 Molarity (M) 6
  • PBS (Gibco; 21600-096)
  • PBS 0.1 % Tergitol (ThermoScientific; 464252500; CAS: 68131-40-8)
  • H2O2 (Chemlab; CL00.2306.1000; CAS: 7722-84-1)
  • MeOH (VWR; 20846.361; CAS: 67-65-1)-
  • Iba1 (Abcam; Ab5076; RRID: AB_2224402]
  • Rabbit serum (Dako; X0902)
  • Rabbit anti-goat biotinylated (Dako; E0466)
  • GFAP (Dako; z0334, RRID: AB_10013382)
  • Goat serum (Dako; X0907)
  • Goat anti-rabbit biotinylated (Abcam; ab6720; RRID: AB_954902)
  • Streptavidin/HRP antibody (Dako; P0397 OR Invitrogen; S911)
  • 3,3'-Diaminobenzidine tetrahydrochloride (DAB) (Sigmaaldrich; D5905; CAS: 7411-49-6)
  • Timer
  • Distilled water (AD)
  • EtOH (Fisher Scientific; E/0650DF/08; CAS:64-17-5)
  • Histoclear II (Agar scientific; A2-0105; CAS Alkyl Hydrocarbons: 64742-48-9; CAS Orange Terpenes: 5989-27-5)
  • DPX (Sigma-Aldrich; 06522; CAS: 84-74-2)
Day 1: ANTIGEN RETRIEVAL
Rinse the sections with PBS (Gibco; 21600-096).
Add 500 µL 1 Mass Percent Citrate buffer (6 ) in each well. Make sure the tissue is flat and completely on contact with the buffer!

00:30:00 incubation at 80 °C , 00:20:00 in the same buffer On ice .

50m
1X PBS Rinse.
1X PBS Rinse.
1X PBS 00:05:00 on the wobbler.

5m
Day 1: QUENCH ENDOGENOUS PEROXIDASE ACTIVITY
3 % H2O2 (Chemlab; CL00.2306.1000; CAS: 7722-84-1) + 10 % MeOH (VWR; 20846.361; CAS: 67-65-1)-PBS is mixed in the hood. 500 µL per well, incubated 00:10:00 at Room temperature on the wobbler.
Note
Example: 7 ml of PBS 3 % H2O2 + 10 % MeOH: 5.6 ml PBS + 0.7 ml 30 % H2O2 + 0.7 ml of 100 % MeOH.




10m
1X PBS 0.1% Tergitol (Thermo Scientific; 464252500; CAS: 68131-40-8) (PBS-T)
1X PBS-T 0.1 % Tergitol 00:05:00 on the wobbler.

5m
1X PBS-T 0.1 % Tergitol 00:05:00 on the wobbler.

5m
Day 1: PRIMARY ANTIBODY
Add 250 µL per well and let it incubate Overnight at Room temperature .
For Iba1: Add Iba1 1:500 (Abcam; Ab5076; RRID: AB_2224402) in PBS + 10 % rabbit serum (Agilent; X0902)
For Gfap: Add Gfap 1:2000 (Agilent; z0334, RRID: AB_10013382) in PBS + 10 % goat serum (Agilent; X0907).

5m
Day 2
1 x PBS-T rinse.
1 x PBS-T 00:05:00 on wobbler.

5m
1 x PBS-T 00:05:00 on wobbler.

5m
Day 2: SECONDARY ANTIBODY
Add 250 µL /well and let it incubate 00:30:00 at Room temperature on the wobbler.
Iba1: Add rabbit anti-goat biotinylated (Agilent; E0466) 1:600 in PBS-T.
Gfap: Add goat anti-rabbit biotinylated 1:600 (Abcam; ab6720; RRID: AB_954902) in PBS-T.

30m
1 x PBS-T rinse.
1 x PBS-T 00:05:00 on wobbler.

5m
1 x PBS-T 00:05:00 on wobbler.

5m
Day 2: STR-HRP
Streptavidin/HRP antibody (Agilent; P0397 OR Invitrogen; S911) is diluted 1:1000 in PBS-T.
Note
Example: 6.5 ml: 6.5 µl STR-HRP + 6500 µl PBS-T 0.1%

Add 250 µL /well and let in incubate 00:30:00 at Room temperature on the wobbler.

30m
1 x PBS-T rinse.
1 x PBS-T 00:05:00 on wobbler.

5m
1 x PBS-T 00:05:00 on wobbler.

5m
Day 2: DAB

Note
!! work in the hood during the whole DAB step!!
Wear double gloves and an extra pair of covering sleeves.
Take with you:
  • 25 mL PBS pH 7.6 in a 50 ml falcon covered with aluminium foil.
  • PBS-T to rinse
  • 1 tablet of 10 mg 3,3'-Diaminobenzidine tetrahydrochloride (DAB) (Sigma; D5905; CAS: 7411-49-6)
  • An Eppendorf tube with H2O2 (you need 7 µl)
  • 0.22 µm filter (Carl Roth; SE2M035I07)
  • 10 ml syringe
  • Plastic bag
  • Extra prepared plates with the same label as the ones you are using now.


Dissolve the DAB tablet in 25 mL PBS and cover the tube with aluminium foil.

After dissolving most of the DAB, the solution is filtered using the 0.22 µm filter and the 10 ml syringe.
Add 7 µL H2O2.

Remove PBS-T from the wells.
Add 250 µL /well of the DAB solution and let it react for a few minutes at Room temperature
Iba1: 00:06:00 reaction time after starting pipetting the first well.
GFAP: 00:03:00 reaction time after all 24 wells were pipetted.

9m
PBS-T rinse.
PBS-T rinse.
Fill the new plate with PBS and move the sections to the new plate.
Rinse the sections with ½ PBS + ½ AD in a petri dish.
Mount the sections on the pork gelatine coated glasses and let them dry Overnight .

5m
Day 3: DEHYDRATION
30m
  • 00:05:00 70 % EtOH
  • 00:05:00 90 % EtOH
  • 00:05:00 100 % EtOH (Fisher Scientific; E/0650DF/08; CAS:64-17-5)
  • 00:05:00 100 % EtOH
  • 00:05:00 Histoclear II (Agar scientific; A2-0105; CAS Alkyl Hydrocarbons: 64742-48-9; CAS Orange Terpenes: 5989-27-5)




25m
Cover with #1.5 cover slips (epredia; BB02400500SC13MNZ0) by using DPX (Sigma-Aldrich; 06522; CAS: 84-74-2). To put DPX use a Pasteur pipet as a “spoon” since the solution is very viscous. Let them dry Overnight in the hood.

5m
Day 4: VISUALIZATION AND QUANTIFICATION
Visualise the slides with the Leica aperio CS2 slide scanner at 20x magnification.
Quantify the images via Fiji.
Protocol references
This protocol is kindly shared by María Sanchiz Calvo and Eduard Bentea of the Prof. Veerle Baekelandt lab.