Mar 01, 2022
Diaminobenzidine (DAB) Staining
- 1University of Ottawa
Protocol Citation: Haley Geertsma 2022. Diaminobenzidine (DAB) Staining. protocols.io https://dx.doi.org/10.17504/protocols.io.b5s9q6h6
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: March 01, 2022
Last Modified: March 01, 2022
Protocol Integer ID: 58945
Abstract
This protocol is used to stain paraffin-embedded mouse brain tissue.
To paraffin-embedded tissue, deparaffinize in 100% xylenes for 3x 5 minutes.
15m
Incubate in 100% ethanol for 2x 5 minutes.
10m
Incubate in 70% ethanol for 5 minutes.
5m
Incubate in 50% ethanol for 5 minutes.
5m
Incubate in 1X PBS for 5 minutes.
5m
Perform antigen retrieval by incubating tissue in 1X sodium citrate for 20 minutes at 95oC.
Sodium citrate: 10mM sodium citrate + 0.05% Tween-20, pH6
20m
Wash with 1X PBS for 5 minutes.
5m
Incubate in 0.9% H2O2 for 10 minutes at room temperature.
10m
Wash with 1X PBS for 5 minutes.
5m
Incubate in blocking buffer for 1 hour at room temperature.
Blocking buffer: 10% serum + 0.05% Triton X-100 in 1X PBS
1h
Wash with 1X PBS for 5 minutes.
5m
Incubate in primary antibody diluted in blocking buffer overnight at 4oC.
1d
Wash with 1X PBS for 5x 5 minutes.
30m
Incubate in secondary antibody diluted in blocking buffer for 1 hour at room temperature.
1h
Wash with 1X PBS for 5x 5 minutes.
30m
Incubate in streptavidin horseradish peroxidase diluted in 1X PBS for 1 hour at room temperature.
1h
Wash with 1X PBS for 5x 5 minutes.
30m
Expose to DAB for 3-10 minutes then rinse in 1X PBS for 2x 2 minutes.
15m
Incubate in 50% ethanol for 2 minutes.
2m
Incubate in 70% ethanol for 2 minutes.
2m
Incubate in 100% ethanol for 2 minutes.
2m
Incubate in 100% xylenes for 5 minutes.
5m
Coverslip with Permount and a #1.5 coverslip.
1m