Background:

3,3/-Diaminobenzidine (DAB) is the most commonly used substrate for demonstrating the presence of horseradish peroxidase in immunohistochemistry or immunoblotting. It produces a stable water/alcohol insoluble reddish brown product. This effect is enhanced by modifying the DAB solution with the addition of metal ions to produce a more dense reaction forming a localized deep blue/black precipitate.1,2 Leinco's Metal enhanced DAB Substrate Kit is a stable solution developed to give enhanced color reaction with low background. The reagents supplied are sufficient for making 300ml working solution.

Components:

1. 6 ml Buffer Stock Solution (Storage 4°C)
2. 12 ml Solutle DAB Stock Solution (Storage -20°C)
3. 6 ml Hydrogen Peroxide Stock Solution (Storage 4°C)
4. 6 ml Enhancing Stock solution (Storage 4°C)

Hazard Description:

Warning: As DAB and nickel are suspected carcinogens care should be taken in handling and disposal of the working solution. Gloves, lab coats and good laboratory procedures are necessary when using the reagents. For disposal the working solution is discarded in 3% potassium permanganate (KMNO₄) and 2% sodium carbonate in distilled water. Wash out all containers used for substrate staining with this solution. Dispose according to local regulations.

Direction For Product Use:

FOR TISSUE SECTIONS
1. Add the working solution of DAB and incubate at room temperature for 15 seconds or until color develops.
2. Wash off excess DAB under running water for 5 mins.
3. Counterstain and mount in aqueous or dry medium.

FOR IMMUNOBLOTTING
1. Add the working solution of DAB and incubate at room temperature for 15 seconds or until color develops.
2. Remove excess DAB with two washes in distilled water for 10 mins.
3. Air dry and store in a dark place.

FOR DOUBLE STAINING
Note: It is important to stain the tissue for the first antigen with the metal enhancement.
1. Process the tissue section for labeling the first antigen with peroxidase.
2. Stain with DAB Substrate Kit with metal enhancement.
3. Wash well in distilled water for 5 minutes.
4. Process the tissue section for labeling second antigen with peroxidase.
5. Stain with DAB Substrate kit without metal enhancement.
6. Wash off excess DAB under running water.
7. Counterstain and mount in aqueous or dry medium.

DAB without enhancement will produce a reddish brown precipitate and the Metal Enhancing solution will produce a dark blue/black colored precipitate.