Mar 14, 2024
DAB Staining
- daniel.dautan daniel1,2,
- Per Svenningsson1,2
- 1Department of Clinical Neuroscience, Karolinska Institutet, 171 76 Stockholm, Sweden;
- 2Aligning Science Across Parkinson’s (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: daniel.dautan daniel, Per Svenningsson 2024. DAB Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj34zplk5/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: February 06, 2024
Last Modified: September 23, 2024
Protocol Integer ID: 94763
Keywords: ASAPCRN, DAB, immunohistochemistry
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: 020608
Abstract
3, 3'-diaminobenzidine (DAB) staining of mouse brain tissue
Guidelines
All steps should be done on an orbital shaker.
Materials
Antibodies:
- anti-Tyrosine Hydroxylase (ab152), dilution 1:500
- Biotin-conjugated goat anti rabbit secondary antibody, dilution 1:300
Reagents:
- 1x PBS
- 30% H202
- Methanol
- Primary TH: AB 152, Millipore, NFAB152
- Secondary: anti-rabbit lgG (whole molecule) Biotin Conjugate, Sigma B-6648
- DPX mounting medium, Sigma Aldrich, https://www.sigmaaldrich.com/cata1og/product/SIGMA/06522?lang=en®ion=SE
- Antigen retrieval: Tris-EDTA, pH 9.0 or Sodium citrate, pH=6.0, 80°
- Tris-EDTA buffer (lOmM Tris Base, 1 mM EDTA, 0.05% Tween 20, pH 8.0): Mix 1.21 g Tris Base, 0.37 g of disodium EDTA in 1000 ml of distilled water. Adjust the pH to 9.0 with 1N Na OH and then add 0.5 ml of Tween 20. Store at RT for up to 3 months; for extended storage, store at 4 °C.
- Sodium citrate buffer: Tri-sodium citrate (dihydrate) 2.94 g, Distilled water 1 L. Mix to dissolve. Adjust pH to 6.0 with 1N HCI. Add 0.5 ml Tween 20 and mix well. Store at RT for 3 months or at 4°C for longer storage.
Safety warnings
The DAB solution is highly toxic and should be discarded properly according to institutional guidelines.
Staining/Mounting Process
Staining/Mounting Process
Wash freshly sectioned slices of tissue 2-3 times with 1X PBS to remove OCT.
Quench sections for 00:15:00 in 3 mL of quenching solution (0.1 mL 30% H2O2, 0.1 mL methanol, and 0.8 mL 1X PBS).
15m
Wash 4-5 times in 1X PBS.
Block sections in 2 mL of 5% goat serum in 0.25% T-PBS for 01:00:00 Room temperature .
1h
Stain with primary antibody (pSer129 primary antibody 1:500) overnight at 4 °C . Dilute primary antibody in 2.5% serum in 0.25% T-PBS (1 mL per well).
Wash sections 4-5 times with 1X PBS.
Transferred sections into secondary solution (1 mL of 1% serum in 0.25% T-PBS) for 02:00:00 at Room temperature .
2h
Wash sections 4-5 times in 1X PBS.
Transfer into ABC Kit solution (PK4000, Vector Laboratories) containing 10µl of solution A and 10µl of solution B for 1ml of 1X PBS for 01:00:00 at Room temperature .
1h
Wash sections 4-5 times in 1X PBS.
Transfer sections to the DAB working solution (SK-4100, Vector Laboratories) under a fume hood. Monitor until it stains well. Well-plate containing sections were gently shaken during staining and the reaction was stopped with transfer to 1X PBS based on dark-signal intensity on the fastest arising staining.
Wash sections 3-5 times in 1X PBS.
Mount sections on microscope slides.
Dry sections Overnight at Room temperature .
1h
Dehydrate sections starting with 2 baths of distilled water (~00:02:00 ).
2m
Wash with 70% ethanol (2 times ~00:02:00 ).
2m
Wash with 95% ethanol (2 times ~00:02:00 ).
2m
Wash with 100% ethanol (2 times ~00:02:00 ).
2m
Wash with 100% xylene (2 times ~00:05:00 ) to allow section dehydration.
5m
Dry sections at Room temperature (~00:05:00 ) and covered with DPX mounting medium.
5m