How does DAB chromogen work?
It is most often used in immunohistochemical (IHC) staining as a chromogen. In DAB staining, DAB is oxidized by hydrogen peroxide in a reaction typically catalyzed by horseradish peroxidase (HRP). The oxidized DAB forms a brown precipitate, at the location of the HRP, which can be visualized using light microscopy.
What is DAB staining?
Diaminobenzidine is oxidized by hydrogen peroxide in the presence of hemoglobin to give a dark-brown color. This color change is used to detect fingerprints in blood. The solubility of DAB in water allows for adaptability compared to other detection solutions which use toxic solvents.
How do I prepare for IHC DAB?
1% DAB (20x) in Distilled Water: Add 0.1g of DAB (3,3′-diaminobenzidine, Sigma Cat#D8001 or DAB-tetrahydrocholoride) in 10 ml distilled water. Add 10N HCl 3-5 drops and solution turns light brown color. Shake for 10 minutes and DAB should dissolve completely.
What is HRP substrate?
Horseradish peroxidase (HRP) substrates are used in the immunostaining and immunodetection of specific proteins and antigens. HRP is a widely used label, often conjugated to secondary antibodies in immunochemical applications such as immunohistochemistry, western blotting, and ELISA.
How do you stop the DAB reaction?
10- Wash away DAB to stop reaction….As a general protocol you can try the following:
- Wash with PBS 3 x 5 min.
- Endogenous peroxidase blocking 1% H2O2 in PBS 10 min.
- Wash with PBS 3 x 5 min.
- Blocking with 10% BSA60 min @ 37 ̊C.
- Primary antibody in PBS-T/1% BSA 60 min @ 37 ̊C.
- Wash with PBS 3 x 5 min.
How do HRP and DAB interact?
HRP catalyzes the conversion of chromogenic substrates (e.g., TMB, DAB, ABTS) into colored products, and produces light when acting on chemiluminescent substrates (e.g. Enhanced Chemiluminescence by luminol).
What is DAB used for?
Dabs or dabbing are the names for the use of concentrated butane hash oil (or BHO). It is a relatively new method of administering/ingesting cannabis that involves the inhalation of highly concentrated tetrahydrocannabinol (THC), the main active chemical in cannabis.
How do you increase DAB stain?
The best way to enhance the DAB reaction is during the reaction itself, since it is the HRP enzyme that uses the DAB, along with the H2O2, to create the colored product. So it doesn’t matter if you are doing immunohistochemistry, detecting neurobiotin in slices, or Western Blots, as long as you have active HRP.
What is the purpose of DAB in IHC?
DAB (3, 3′-diaminobenzidine) produces a dark brown reaction product and can be used for both immunohistochemical and blotting applications. DAB is effective as a single label or as a second color for multiple antigen labeling. Because of its heat-resistance, DAB can be used in IHC/ ISH double labeling applications.
Why does TMB turn blue?
TMB turns blue in presence of H2O2 and Peroxidase. Sometimes metals also act as peroxidase mimic. Just avoid any peroxide and peroxidase in proximity. You should consider preparing a fresh stock.
What color does HRP produce?
Colorimetric substrates for ELISA applications
| Substrate | Enzyme | Absorbance and color |
|---|---|---|
| OPD Substrate | HRP | 490 nm (450 nm) Green (Orange) |
| PNPP (p-Nitrophenyl Phosphate) | ||
| 1-Step PNPP Substrate Solution | AP | 405 nm Yellow |
| PNPP Substrate | AP | 405 nm Yellow |