Intended use: DAB Substrate kit High Contrast is developed for immunohistochemical and in situ-hybridisation staining procedures with horse radish peroxidase (HRP). DAB (3,3’-Diaminobenzidine) leads to the formation of a brown precipitate at the location of the target antigen or target nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy. The kit is especially useful when a high contrast between chromogen and counter stain is desired. Compared to standard DAB staining systems the DAB Substrate High Contrast kit gives a darker brown colour and a higher sensitivity.
Reagents provided: 30 ml DAB Chromogen (liquid DAB concentrate)500 ml DAB Substrate Buffer High Contrast
Storage and handling: The solutions should be stored at 2-8°C without fur ther dilution. Please store the reagents in a dark place and do not freeze them. Under these conditions the solutions are stable up to the expiry date indicated on the label. Do not use product after the expiry date. The working solution should be prepared freshly at the day of use. Once the two reagents are combined, the resulting solution is stable for up to six hours. Excess working solution should be disposed as hazardous substance. A positive and a negative control have to be carried out in parallel to the test material. If you observe unusual staining or other deviations from the expected results which could possibly be caused by the kit reagents please contact our technical support.
Reagent preparation: Add 50 µl of DAB Chromogen (DAB concentrate) to 1 ml of DAB Substrate Buffer High Contrast and mix thoroughly. Note: Typical working concentrations are 50 µl (0.9 mg) DAB per ml substrate buffer. The colour intensity can be adjusted by decreasing or increasing the DAB concentration in the working solution. Maximum sensitivity in immunohistochemical staining can be achieved by working concentrations of about 80 µl (1.5 mg) DAB per ml substrate buffer.
Procedure: 1) Rinse the slide with wash buffer after the previous incubation step. 2) Apply the DAB High contrast working solution to the slide. Incubate for 5-15 minutes. 3) Rinse with distilled H2O. 4) Counterstain with haematoxylin for about 30 seconds up to 5 minutes (depending on the desired staining intensity). 5) Rinse with distilled H2O. 6) Blueing in tap water for at least 5 minutes. 7) Dehydrate through a graded series of ethanol and clear in xylene. Mount with a permanent mounting medium. Note: It is also possible to mount DAB High Contrast with aqueous mounting media.
Expected results: During the reaction of the substrate with horse radish peroxidase in presence of the chromogen DAB, a brown precipitate is formed at the location of the target antigen or nucleic acid. The precipitate is insoluble in aqueous and organic solvents and can be observed by light microscopy.
References: Elias JM Immunohistopathology – A practical Approach to Diagnosis ASCP Press 2003/Nadji M and Morales AR Ann N.Y. Acad Sci 420:134-139, 1983
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