DAB is one of the most commonly used oxidative chromogenic dyes for the detection of peroxidase in immunohistochemistry. In the presence of H2O2 and peroxidase, DAB is oxidized into a brown pigment. These brown pigments are aniline black type compounds and are firmly deposited around peroxidase on cell membranes or tissues. Most DAB available on the market appears brown as a result of oxidation during processing or storage. On the other hand, Dojindo offers a high quality DAB with a white or slightly reddish-gray powder appearance that is good for sharp staining.
Preparation of Sample Staining Solution
1. Dissolve 9 mg DAB with 1 ml PBS to prepare 100X DAB solution.
2. Mix 5 μl of 30% hydrogen peroxidase solution with 1 ml PBS to prepare 200X H2O2 solution.
3. Add 10 ul of 100X DAB solution and 5 ul of 200X H2O2 solution to 1 ml PBS to prepare staining solutiona).
4. Add the staining solution to a sample in a staining chamber, and incubate the sample at room temperature for 5 minutes to 1 hourb).
5. Wash the sample with PBS several times to stop the staining reaction.
a)The staining solution is not stable. Prepare fresh solution prior to use.
b)For better staining, incubate the sample with 0.09 mg DAB/PBS solution for 10 minutes before adding the staining solution.
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