DAB (3,3’-diaminobenzidine) is the most commonly used amine substrate for histochemical detection of cell surface and intracellular antigens on frozen or embedded tissue sections, cytospins and touchdown preparations. Horseradish peroxidase (HRP), which is bound to the primary or secondary antibody, catalyzes the oxidation of DAB in the presence of peroxidase. The oxidation product of DAB polymerizes to form a brown, sharply localized product, which can be easily detected by light microscopy. Treatment of the DAB product with different metal ions, either darken the reaction (cobalt chloride) or change the color to black (nickel chloride) or blue (cobalt chloride/nickel ammonium sulfate). Staining with osmium tetroxide allows the detection of DAB-labeled product in electron microscopy.
KPL’s optimized DAB Reagent Set includes three convenient drop-bottles: a concentrated DAB solution (25 mg/ml), a concentrated Tris buffer solution (0.1 M) and a peroxidase solution (0.5%). Each kit contains sufficient reagents for staining 500 slides. The kit is stable for one year when stored at 2-8°C. Before staining, the reagents should be equilibrated to room temperature.
We have routinely used the DAB Reagent Set to stain mouse frozen tissue sections. Before staining, the sections were first dried at room temperature, fixed either in 2% paraformaldehyde, cold acetone and/or methanol (5 min) and washed in PBS (10 mM phosphate buffered saline, pH 7.4; 5min) or used without fixation. The endogenous horseradish peroxidase was quenched by immersing the sections in a 0.3% hydrogen peroxidase/0.3% horse serum/PBS solution for 5 min followed by 2 x 5 min washes in PBS. The slide around the section was dried with a tissue, and the section area circled with a Pap pen to limit the amount of the antibody solution needed. After blocking the tissue with species-specific serum (i.e. same species in which the secondary antibody was raised), the primary antibody was applied and incubated overnight at 4°C. The section was washed again in PBS (2 x 5min) and the secondary HRP- or biotin-labeled antibody was applied and incubated for 1 h. When a biotinylated antibody was used for enhancement of antigen detection, the section was washed 5 min in PBS and incubated with a streptavidin/HRP solution (Vectastain Elite ABC Kit, Vector Laboratories) for 30 min. For detection, the DAB solution was prepared during the last washes (2 x 5 min) with PBS. To prepare the DAB solution, to 5 ml of ddH2O, add 3 drops of Tris buffer (ca. 150 µl), 2 drops of DAB solution (ca. 100 µl) and 2 drops of peroxide solution, mix and keep in the dark. The DAB solution was applied to the section and incubated for 3-5 min under a cover of aluminum foil. The development of the brown color was then observed under a microscope and immediately stopped by removing the DAB (into a chemical hazard waste container) and rinsing the section with dH2O for at least 1 min. (I used the dH2O water coming from the faucet to thoroughly wash). Insufficient washing with dH2O resulted in dark brown staining and an enhanced light brown background. The sections were counterstained with hematoxylin and mounted with xylene. Since the DAB staining enhanced during the hematoxylin counterstain/mounting procedure, I stopped the DAB development when a light brown color was visible. For weak signals, I used enhancement with nickel chloride (50 µl of 80mg/ml), but stopped the development reaction immediately when the color was becoming visible in order to avoid over-staining/background. To control non-specific background staining, I used a slide that was not exposed to the primary antibody.
The KPL DAB detection system is a sensitive, easy to use and reliable detection system for immunohistochemical staining of sections. It has always worked well for me. The intensity of staining can be easily adjusted as desired by observing the color development under a light microscope. Since the DAB is provided in a drop bottle, handling of the hazardous chemical is limited. The package insert provides trouble shooting information, protocols for the staining procedure and buffer preparations.