3,3 -diaminobenzidine chromogen

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. 

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. 

Fig. 12.4. Bromodeoxyuridine (BRDU) labeling of cells in S phase. Routine paraffin sections from mice injected 1 h prior to euthanasia with BRDU have brown nuclei when labeled by immunohistochemistry using diaminobenzidine as a chromogen. These cells were synthesizing DNA at the time of necropsy and therefore incorporated the label. Counting the number of positive nuclei in basal cells per 1000 basal cells yields the proliferation rate. The boxed area in A is enlarged in B to illustrate the large number of positive (dark) nuclei in the skin of an adult chronic proliferative dermatitis mutant mouse (Sharpincpdm/Sharpincpdm).

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 preference for any of the different chromogenic H-donors depends largely on individual laboratories. In general, the most commonly used donors are quite satisfactory. In EIA both the substrate and the product should have an adequate solubility to keep light scattering at a minimum. In contrast, in immunoblotting and EIH 4-chloro-l-naphthol (Chapter 16) and 3,3 -diaminobenzidine (Chapter 17) are preferred as substrate, yielding an insoluble product which precipitates at the location of the enzyme. The most commonly used H-donors in EIA are 5-aminosalicylic acid (5-AS), o-dianisidine... 

Figure 2.3-1 Immunohostochemistry of mouse thymus double staining of cytokera-tin/CD3 lymphocytes and CD4/CD8 lymphocytes. Peroxidase (PO) and alkaline phosphatase (AP) were used as the label for the secondary antibody. PO was visualized by H2O2 and diaminobenzidine, while AP by AS-MX phosphate and fast blue as the substrate and chromogen, respectively. DP double positive SP single positive.

DAB DAB (3,3 -diaminobenzidine tetra-hydrochloride, C12H14N4.4HCI) gives a brown hydrophobic end-product, resistant to alcohol and xylene. DAB chromogen seems to be the most economical, reliable and reproducible chromogen in the immunohistochemistry. 

Fig. 2. Representative examples of the appearance of /ocZ-positive cells in the central nervous system at the light and electron microscopic levels. (A) Representative examples of histochemically stained lacZ-positive neurons in the rat cerebral cortex resulting from an injection of retrovirus into the cerebral ventricles of the embryonic d 16 rat telencephalon. Note that the cell bodies and proximal dendrites are intensely stained (modified with pamission from Luskin et al., 1993). (B) Representative example of an immunohistochemicaUy stained neuron in the olfactory bulb resulting Irom a perinatal injection of retrovirus. The IdcZ-positive cell was revealed using a primary antibody to P-galactosidase diaminobenzidine was used as a chromogen to visualize the secondary antibody conjugated to horseradish peroxidase. In most instances immunohistochemistry can be used to label the fine processes of lacZ-positive cells (modified with permission from Luskin, 1993). (C) An electron micrograph showing a ZacZ-positive (left) and unlabeled (right) neuron in the visual cortex from a rat that received an intraventricular injection of retrovirus at E16. The nucleus and nuclear membrane are conspicuously stained and in the cytoplasm the reaction product is associated preferentially with the endoplasmic reticulum, which extends into the apical dendrite (large arrows) and basal dendrite (arrowhead) of the labeled cell (modified with permission from Luskin et al., 1993).

This is the enzyme reporter of choice in most situations. When used with the chromogen 3.3 -diaminobenzidine (DAB) (32), it yields a brown reaction product that is extremely well localized and insoluble in all the commonly used dehydrating and clearing agents. Slides can thus be permanently mounted in resinous mountant allowing veiy good optical resolution under the microscope. 

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