Diaminobenzidine (DAB) Enhanced Chromogen Kit
The use of 3-3’ diaminobenzidine tetrahydrochloride (DAB) as a chromogen, when performing peroxidase-based immunocytochemical procedures, provides the means of organic solvent-stable permanent chromogen depositions. Therefore, specimens developed with DAB can be dehydrated and mounted with organic solvents-based plastic polymers such as Permount or ClariØn (Cat. No. M05 and M06). Since DAB is a suspected carcinogen, many laboratories avoid the handling of the powdered chemical. However, the availability of DAB tablets minimizes the risk of exposure and facilitates its use in the immunohistology laboratory.
Biømeda's Diaminobenzidine Enhanced Chromogen is significantly more sensitive than the regular DAB.
Nickel ions included in the concentrated buffer formulation intensify the regular DAB color. The DAB Metal depositions are dark brown in color and can be counterstained with alcohol-based hematoxylin or with Biømeda’s Aqueous Hematoxylin (Cat. No. M10). Nuclear Fast Red or Methylene Green can be also used as counterstains.*
These products are designed to produce a color deposition on tissue sections processed by immunocytochemical techniques using alkaline phosphatase or peroxidase as tracers. When using these products, it is very important to select the appropriate chromogen substrate for the enzyme tracer being used in the assay.
One mL of final working solution will yield approximately 5-6 tissue sections. The number of tests performed will vary depending on the application. Estimations are derived from use with the capillary-action based MicroProbe™ system, which draws 150-200 mL per test.
Each 20-tablet kit contains sufficient reagents to make 100 mL of final working solution. The 100 tablet chromogen-only kit will yield 500 mL of final working solution. All other kits contain sufficient reagents to make 300 mL of final working solution.
Refrigerate at 4°C. Do not freeze.
*When using Nuclear Fast Red or Methylene Green, dehydrating the tissue sections and mounting with ClariØn or another organic based mounting medium, is required. Aqueous mounting media like Crystal/Mount are not compatible with these counterstains.
Working Chromogen Preparation: Remove the DAB Enhanced Chromogen kit from the refrigerator 20 minutes before the preparation of the working chromogen solution. This will minimize the condensation of ambient water on the tablets during handling.
1. Transfer 5 mL of distilled water into supplied plastic bottle.
2. Add 5 drops of Concentrated Buffer Reagent. Mix.
3. Add one chromogen tablet. Allow tablet to dissolve for approximately 5 minutes with occasional mixing. Secure the cap of the bottle containing the unused tablets and return it to the original plastic box.
4. After tablet dissolves, add one drop of Concentrated Substrate. Mix well.
5. Apply this solution to the specimens. Make sure to wash the specimens extensively with buffer to remove tracing reagent containing peroxidase.
6. Incubate for 5-10 minutes at room temperature. Wash specimens with distilled water and proceed with the counterstaining steps.
Use the working chromogen solution within 2 hours from the time of the addition of the tablet. Discard unused reagent according to local and state regulations.
DAB is a suspected carcinogen. Avoid contact with skin and clothes.