DAB Substrate (3, 3’-diaminobenzidine) produces a brown reaction product in the presence of peroxidase (HRP) enzyme. Substrate Kit contains all of the reagents necessary toprepare. These reagents are supplied in convenient dropper bottles.
 
REAGENTS
Content
 
Size
Storage
Reagents A
DAB   20×concentrated solution
3ml
-20℃
Reagents B
H2O2  20×concentrated solution
3ml
4-8℃
Reagents C
TBS   20× concentrated solution
3ml
4-8℃
 
STORAGE
Store reagents in original bottles at the required conditions (one year)
Avoid storing reagents or working solution in strong direct light.
 
PREPARATION OF SUBSTRATE WORKING SOLUTION
To 1.0 ml of distilled water
Add 50 µl of Reagents C
Add 50 µl of Reagents A
Add 50 µl of Reagents B
Mix well before use
 
INSTRUCTIONS FOR USE
For Tissues or Cells After incubation with a peroxidase (HRP) detection system, rinse well. Incubate with the substrate working solution at room temperature for 2-10 minutes. Optimal development times should be determined by the investigator. Wash for 5 minutes in water.
 
NOTES
We recommend using glass-distilled water in the preparation of substrate buffer. Deionized water may con-tain inhibitors of the peroxidase reaction. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate.
DAB are carcinogens. Appropriate care should be exercised when using these reagents including gloves, eye protection, lab coats, and good laboratory procedures.