Diaminobenzidine, reaction

MuUink H, Vos W, Jiwa M, Horstman A, der Valk van P, Walboomers JM, Meijer CJ. 1992. Application and comparison of silver intensification methods for the diaminoben-zidine and diaminobenzidine-nickel endproduct of the peroxidation reaction in immunohistochemistry and in situ hybridization. J Histochem Cytochem 40 495-504. 

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. 

The technique described here is for use with monoclonal primary antibodies of mouse origin, but can easily be adapted for use with polyclonal antibodies from other species (i.e., rabbit). This method uses a secondary biotin-labeled antibody and a detection system that employs a biotin-avidin horseradish peroxidase complex linker step, the so-called ABC (avidin-biotin complex) detection system (5) (see Chapter 25). In this detection system, avidin acts as a bridge between the biotinylated secondary antibody and a biotin-labeled peroxidase enzyme. The anchored enzyme, in the presence of H2O2 can then convert the substrate, diaminobenzidine, to a brown or black reaction product that is easily identifiable in the tissue section. 

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. 

Fig. 8. Morphological changes of apoptotic eosinophils induced by dexamethasone (Z2). After eosinophils were treated (a) without or (b) with dexamethasone (2 /u,M) for 12 h, cells were harvested and detected by TUNEL assay using the In Situ Cell Death Detection Kit (Boehringer Mannheim). Briefly, cells were fixed with 4% paraformaldehyde and permeabilized by proteinase K and incubated with the TUNEL reaction mixture containing terminal deoxynucleotidyl transferase (TdT). After washing to remove unbound enzyme conjugated antibody, the horseradish peroxidase retained in the immune complex was visualized by a substrate reaction with diaminobenzidine. The cell nucleus was counterstained with methanol green. Apoptotic eosinophils with nuclear DNA breaks were seen to stain dark brown using a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan) in Fig. 8b.

The technique described above is not useful for removing colored peroxidase reaction products (e.g., diaminobenzidine oxidation products) from blots Thus, we avoid detection methods based on these reactions. On the other hand, peroxidase-based luminescent assays (20) do not deposit a chemical reaction product on the blot and are compatible with this erasure method (21). An example of the use of this erasure method (Section 3 3, steps 1-5) after chemiluminescent detection is shown in Fig 2. 

Yamaguchi, I., Osakada, K., Yamamoto, T., Polyrotaxane containing a blocking group in every structural unit of the polymer chain. Direct synthesis of poly(alkylenebenzimidazole) rotaxane from Ru complex-catalyzed reaction of 1,12-dodecanediol and 3,3-diaminobenzidine in the presence of cyclodextrin. J. Am. Chem. Soc. 1996, 118, 1811-1812. 

In keeping with current trends in immunohistochemistry to develop alternatives to biotin-streptavidin detection methods, a fluorescyl-tyramide amplification system has recently been introduced (FT-CSA). In this procedure peroxidase is associated with a tissue-bound primary antibody by application of a secondary antimouse Ig antibody to which peroxidase has been conjugated. The peroxidase catalyzes the conversion and deposition of fluorescyl-tyramide onto the tissue section. At this point the reaction can be terminated and viewed by fluorescence microscopy, or the signal can be converted to a colorimetric reaction by the sequential application of an anti-fluorsecein antibody conjugated to peroxidase followed by a diaminobenzidine-hydrogen peroxide substrate. 

Around 1960 Carl Marvel at Illinois discovered that the reaction between 3,3, 4,4 -tetraaminobiphenyl (diaminobenzidine) (186) and diphenyl isophthalate yielded the strong, stable poly benzimidazoles. The thermal and chemical stability of their fibers make them suitable for parachutes, space suits and firefighting clothing. Several novel polymerizations were reported, including with 1,2,5,6-tetraaminoanthraquinones. 

Fig. 2. Distribution of glutaminase-like immunoreactivity in the central nervous system. The rat brains were fixed and immunostained as reported before (Kaneko et al., 1989). Briefly, rats were fixed at room temperature by transcardial perfusion of 0.2% formaldehyde, 75%-saturated picric acid and 0.1 M sodium-phosphate, pH 7.0, and the brain blocks were further placed for 3 days at 4°C in 2% formaldehyde, 75%-saturated picric acid and 0.1 M sodium-phosphate, pH 7.0. The brain sections (30 p.m thick) were incubated with 10 p.g/ml monoclonal anti-glutaminase IgM, MAb-120, then with 10 p.g/ml biotinylated goat anti-glutaminase antibody (Vector), and finally with avidin-biotinylated peroxidase complex (ABC Vector), and the bound peroxidase was developed brown by reaction for 10-30 min with 0.02% diaminobenzidine-4HCl (DAB), 0.003% H2O2 and 50 mM Tris-HCl, pH 7.6. (o) Upper cervical cord.

Fig. 7.9. General catalytic reaction mechanism of POase. Note that excess substrate leads to enzyme inactivation. Hydrogen donors such as 3,3 -diaminobenzidine (DAB), 4-chloro-l-naphthol, 3,3, 5,5 -tetramethylbenzidine (TMB) and o-dianisidine (ODA) are often used, but the color reaction (III) proposed by Conyers and Kidwell (1991) yields superior results.

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