Aug 01, 2024
DAB Immunohistochemistry (IHC) Staining for Stereological Analysis
- 1Department of Pharmacology and Physiology, Faculty of Medicine, Université de Montréal;
- 2Department of Neurosciences, Faculty of Medicine, Université de Montréal
Protocol Citation: Nicolas Giguère, louis-eric.trudeau Trudeau 2024. DAB Immunohistochemistry (IHC) Staining for Stereological Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2lyw1jmvx9/v1
Manuscript citation:
Adoptive transfer of mitochondrial antigen-specific CD8+ T-cells in mice causes parkinsonism and compromises the dopamine system
MN Elemeery, A Tchung, S Boulet, S Mukherjee, N Giguère, J-F Daudelin, A Even, R Hétu-Arbour, D Matheoud, JA Stratton, N Labrecque, L-E Trudeau
bioRxiv 2024.02.26.582098; doi: https://doi.org/10.1101/2024.02.26.582098
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 29, 2024
Last Modified: August 01, 2024
Protocol Integer ID: 104423
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's (ASAP)
Grant ID: ASAP-000525
Abstract
DAB (3,3'-diaminobenzidine) is oxidized in the presence of peroxidase and hydrogen peroxide resulting in the deposition of a brown, alcohol-insoluble precipitate at the site of enzymatic activity.DAB (3, 3'-diaminobenzidine) produces a dark brown reaction product and can be used for immunohistochemical and applications. This protocol details the DAB immunohistochemistry staining for stereological analysis of 40 µM slices cut with cryostat and stored in antifreeze.
Guidelines
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee
Materials
Reaction mixture:
| A | B | |
| 0.2M acetate buffer | 5mL | |
| Nickel ammonium sulfate | 250mg | |
| β-D-Glucose | 20mg | |
| Ammonium chloride | 4mg |
0.2 M Acetate Buffer pH 6.0:
Prepare 1L of 0.2N acetic acid (11.5 ml glacial acetic acid and top up to 1L with H2Odd)
Mix 900 ml of 0.2M sodium acetate and 51.7 ml
Top up to 1L with H2Odd
Check pH
Glucose oxidase (1mg/ml)
2 mg of glucose oxidase
2 ml of acetate buffer 50 mM
Make aliquots of 80 ul in advance
Keep the powder and aliquots at -20 degrees
Procedures
Procedures
1w 6d 18h 29m 30s
1w 6d 18h 29m 30s
Basic protocol for a peroxidase reaction using free floating sections.
Note
The following steps should be done on a rotating table:
Wash in 0.01 Molarity (M) PBS for 00:10:00 (use cell strainers for all washing steps).
10m
Wash in 0.01 Molarity (M) PBS containing 0.9% H202 for 00:10:00 (90 µL in 10 mL PBS 0.01M) (Blocking of endogenous peroxidase).
10m
Wash in 0.01 Molarity (M) PBS (3x 10 min).
Wash in 0.01 Molarity (M) PBS for 00:10:00 (1/3).
10m
Wash in 0.01 Molarity (M) PBS for 00:10:00 (2/3).
10m
Wash in 0.01 Molarity (M) PBS for 00:10:00 (3/3).
10m
Incubate in primary antibodies for 48:00:00 @ 4 °C under stiring.
- Diluted to its optimal titer (1:1000-1:10,000) in 0.01 Molarity (M) containing 0.3% Triton X-100
Note
* Better to avoid sodium azide because of its blocking action on HRP
• TH Rabbit – 1:1000
• GFP Rabbit 1:5000
• 5-HT Rabbit 1:1000
2d
Wash in 0.01 Molarity (M) PBS (3x 10 min).
Wash in 0.01 Molarity (M) PBS for 00:10:00 (1/3).
10m
Wash in 0.01 Molarity (M) PBS for 00:10:00 (2/3).
10m
Wash in 0.01 Molarity (M) PBS for 00:10:00 (3/3).
10m
Incubate in biotinylated secondary antibodies for 12:00:00 at 4 °C under stiring.
• Diluted to 1:200 in 0.01 M PBS containing 0.3% Trition X-100 (this antibody is stored in 1:2 glycerol, consequently the concentration will be 1:100).
12h
Wash in 0.01 Molarity (M) PBS (3x 10 min).
Wash in 0.01 Molarity (M) PBS for 00:10:00 (1/3).
10m
Wash in 0.01 Molarity (M) PBS for 00:10:00 (2/3).
10m
Wash in 0.01 Molarity (M) PBS for 00:10:00 (3/3).
10m
Incubate in streptavidin horseradish peroxidase for 03:00:00 @ Room temperature under stiring.
• Diluted to 1:200 in 0.01 Molarity (M) PBS containing 0.3% Triton X-100
3h
Wash in 0.01 Molarity (M) PBS (3x 10 min).
Wash in 0.01 Molarity (M) PBS for 00:10:00 (1/3).
10m
Wash in 0.01 Molarity (M) PBS for 00:10:00 (2/3).
10m
Wash in 0.01 Molarity (M) PBS for 00:10:00 (3/3).
10m
Prepare a reaction mixture which contains:
| A | B | |
| 0.2M acetate buffer | 5 mL | |
| Nickel ammonium sulfate | 250 mg | |
| β-D-Glucose | 20 mg | |
| Ammonium chloride | 4 mg |
Note
* Once the nickel ammonium sulfate is dissolved add 5 mL ddH2O.
For 0.2 M Acetate Buffer pH 6.0
- Prepare 1 L of 0.2 Molarity (M) sodium acetate (27.216 g for 1L)
- Prepare 1 L of 0.2 Mass Percent acetic acid (11.5 mL glacial acetic acid and top up to 1L with H2Odd)
- Mix 900 mL of 0.2 Molarity (M) sodium acetate and 51.7 mL of 0.2 Mass Percent acetic acid
- Top up to 1 L with H2O dd.
- Check the pH (Should be 6 ).
Prepare DAB solution (10mg/ml); 1 pill (10 mg) of DAB (-20 °C ) in 1 ml aliquot of ddH2O. Vortex for 00:01:00 until the pill is dissolved.
Note
WARNING: THESE STEPS USE 3.3’ DAB WHICH IS CARCINOGEN. THE RESULTING WASTE MUST BE THROWN IN A BLACK GARBAGE BAG AND THEN IN ETHIDIUM BROMIDE CONTAINER. THE TOOLS TO REUSE, SUCH AS BRUSH, MUST BE CLEANED WITH BLEACH AND H2O.
1m
Prepare Glucose oxidase (1 mg/mL )
Glucose oxidase stock:
- 2 mg of glucose oxidase
- 2 mL of acetate buffer 50 millimolar (mM)
- Make aliquots of 80 µL in advance .
- Keep the powder and aliquots at -20 °C .
Add 250 µL of DAB (10mg/1mL); 1 mL for 10 mL reaction mixture) and 20 µL of glucose oxidase for every 2.5 mL of mixture (1mg/mL); 80 µL for 10 mL reaction mixture) to 10 mL of reaction mixture.
Transfer the DAB reaction mixture in 10 ml syringe with 0.2 µm filter and place 1 mL of DAB reaction mixture per well (Use a 12-wells plate).
Rinse sections in 0.1 Molarity (M) acetate buffer for 00:01:00 .
For 0.1 M Acetate Buffer pH 6.0
- Mix 500 mL of 0.2 Molarity (M) Acetate Buffer and 500 mL of H2Odd.
1m
Transfer sections to multi-wells containing DAB reaction mixture.
Wait 00:00:30 -00:10:00 for sections to develop a dark blue/purple nuclear stain under stirring (for TH, 45s is sufficient)
10m 30s
Remove immediately the sections from DAB reaction mixture.
Wash in 0.1 Molarity (M) acetate buffer for 00:10:00 (it will stop reaction).
10m
Mount sections on charged slides in 0.1M acetate buffer.
Allow slides to dry 48:00:00 -96:00:00 .
Note
(96h is better to avoid slices from coming off).
6d
Defat slides in a series of ethanol baths 50-100% and then 00:02:00 in Xylene.
Note
This step must be done under chemical hood.
• 00:02:00 H2O
• 00:02:00 Cresyl Violet (00:30:00 at 37 °C before staining)
• 00:01:00 H2O
• 00:01:00 H2O
• 00:01:00 EtOH 50%
• 00:01:00 EtOH 70%
• 00:01:00 EtOH 90%
• 10 dips EtOH 90%
• 00:01:00 EtOH 100%
• 00:01:00 Isopropanol
• 00:02:00 Xylene
• 00:02:00 Xylene
47m
Cover with Permount under chemical hood.
Let dry 48:00:00 -72:00:00 under hood.
5d