Diaminobenzidine (DAB) Tablets
The use of 3-3’ diaminobenzidine tetrahydrochloride (DAB) as a chromogen, when using 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 solvent-based plastic polymers such as Permount or ClariØn (Cat. M05 and M06). Since DAB is a suspected carcinogen, many laboratories avoid the handling of the powdered chemical. However, the availability of DAB tableted form minimizes the risk of exposure and facilitates its use in the immunohistology laboratory.
DAB chromogen can be used in membrane-based assays, immunohistochemical and in situ hybridization procedures that employ peroxidase labeled conjugates.Each Biømeda DAB tablet contains 5 mg of DAB tetrachloride and buffer materials to make 5 to 10 mL of buffered DAB solution. After dissolving a tablet it is necessary to add hydrogen peroxide. DAB substrate solution is unstable, therefore it is recommended to prepare fresh working solution for each application.
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 microliters per test.
Each 100-tablet kit contains sufficient reagents to make 500-1000 mL of final working solution.
Refrigerate at 4°C. Do not freeze.
Working Chromogen Preparation:
Remove the DAB tablets 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 to 10 mL of distilled water into the mixing container, add2 drops approx. 100 microliters of 0.5M phosphate buffer, pH7.2.
2.Add one chromogen tablet. Allow tablet to dissolve for approximately 5 minutes with occasional mixing. Secure the cap of the bottle containing the three unused tablets. You may vortex the solution to speed up tablet dissolving.3. After the tablet dissolves, add one drop (approx. 50 microliters) of 3% hydrogen peroxide. Mix well. Working chromogen solutions can be filtered using a syringe filter after this step (optional).4.Apply this solution to the specimens. Make sure to wash the specimens extensively with buffer to remove the tracing reagent containing peroxidase.
5.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 the unused reagent according to local and state regulations.