DAB, Diaminobenzidine

DAB (diaminobenzidine dihydrochloride Sigma, St Louis, MO) stock solution (80X 32 mg/mL in water). This should be handled with care because it is a possible carcinogen. 

Bt, Bacillus thuringiensis DTT, dithiothreitol PMSF, phenylmethylsulfonyl fluoride GST, glutathion-S-transferase PAGE, polyacrylamide gel electrophoresis DIG, digoxigenin HRP, horseradish peroxidase DAB, diaminobenzidine tetrahydrochloride BBMV, brush border membrane vesicle Bti, Bacillus thuringiensis subsp. israelensis... 

Enzymes. Enzymes too are useful labels and several have been employed, with peroxidase and alkaline phosphatase being the most popular to date. One important feature of this technique is for the enzyme to be able to convert a soluble substrate into an insoluble product in order to localize the antigen properly. Several suitable substrates are available with 3 3 -diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) being used with peroxidase and 5-bromo-4-chloro-3-indoylphosphate/nitroblue tetrazolium (BCIP/NBT) with alkaline phosphatase. 

In addition to the many enzyme systems available, there are with each a series of chromogenic substrate solutions that can be used to create different colors and locations of reaction products. For the peroxidase system, there are numerous oxidizable compounds that precipitate as a permanent color. The most common and still widely used is 3,3 diaminobenzidine tetrahydro-chloride (DAB). This compound precipitates to a golden brown color when in solution with peroxidase and hydrogen peroxide. This brown color has many subtleties and readily stands out in a tissue section. With practice, it is possible to differentiate specific from nonspecific staining patterns just by examining the characteristics of the precipitated pigment. This material is also insoluble in alcohol and xylene, and therefore the tissue may be routinely dehydrated and cleared without loss of chromogen. 

Hsu, S. and Soban, E. (1982) Color modification of diaminobenzidine (DAB) precipitation by metallic ions and its application for double immunohistochemis-try. J. Histochem. Cylochem. 30, 1079. 

Chromogen solution 3, 3 diaminobenzidine tetrahydrochloride (DAB) (Sigma St. Louis, MO) 30% hydrogen peroxide (H2O2) see Note 3). 

Wear disposable gloves while handling slides covered with chromogen. Particularly, 3,3-diaminobenzidine (DAB) is a chromogen that is a well-known carcinogen. 

The actual response of monoclonal antibodies with individual cells is usually visualized either directly (typically using fluorescent stains) or indirectly [using the reaction of antibody labeled with horseradish peroxidase (HRP) or other enzymes] with diaminobenzidine (DAB) (or other substrate while using other enzymes) under the microscope or in the flow cytometer. The latter, however, is not employed routinely in CSF immunocytology, although it has an advantage in clinical hematology. 

Chloro-l-naphthol (4-CN) and diaminobenzidine (DAB) are used in Soln. B staining with tetramethylbenzidine (TMB) is done in Soln. G at RT for 10-20 min. 

Develop the peroxidase in 3,3-diaminobenzidine tetrahydrochloride (DAB) solution 3 ml DAB stock solution and 12 drops hydrogen peroxide (30% w/v) are dissolved in 400 ml TBS incubate the specimen with this solution for 10 min (perhaps longer, control with microscope) Wash with tap water... 

Peroxidase substrate (DAB). 2 5 mg of 3,3-diaminobenzidine tetrahydrochlonde dissolved m 5 mL phosphate-buffered saline (PBS). Add 2 pL of 30% hydrogen peroxide (H2O2). DAB powder should be handled only in a fume cupboard. 

Solution of the appropriate substrate for the enzyme chosen (e.g., 1 mg/mL p-nitro-phenyl phosphate in 0.1 Mdiethanolamine-HCl, pH 9.8, for alkaline phosphatase conjugates 3,3, 5,5 -tetramethylbenzidine (TMB) or3,3 -diaminobenzidine (DAB) for horseradish peroxidase conjugates). 

Common chromogen systems currently in use include diaminobenzidine (DAB), 3-amino-9-ethyl-carbazole (AEC), Hanker-Yates reagent, alpha-naphthol pyronin used with peroxidase as substrate fast blue, fast red, BCIP- (5-bromo-4-chloro-3-indolyl phosphate) NBT (nitroblue tetrazolium) used with alkaline phosphatase as substrate tetrazolium, tetranitroblue tetrazolium used with glucose oxidase as substrate, and immunogold with silver enhancement (Leong, 1993 Leong et al, 1997a). 

The third and fourth steps differ according to the modification applied. In the first version, purified rabbit antiserum against peroxidase (anti-PO) reacts with above complex as an antigen with antigenic determinants of anti-rabbit-IgG. Then peroxidase (PO) reacts as antigen with bound anti-PO. In the last step bound peroxidase reacts with diaminobenzidine (DAB) and hydrogen peroxide. The excess of hydrogen-peroxide is detected with the osmium tctroxide. 

Diaminobenzidine (DAB) Cover the secdon with this substrate and incubate the slide for 5 min. The product is brown and is stable in alcohols... 

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