Pierce™ CN/DAB Substrate Kit Thermo Scientific™

Thermo Scientific Pierce CN/DAB Substrate Kit includes substrate and peroxide solutions for combined chloronaphthol- and diaminobenzidine-based detection of horseradish peroxidase (HRP) activity in Western blot and tissue staining methods.

This kit combines two popular chromogenic peroxidase substrate compounds (4-CN and DAB) in a single, stable, 10X stock solution. This CN/DAB solution produces an intense dark black precipitate at sites of bound HRP-conjugated antibodies on probed blots and tissue samples. The Pierce CN/DAB Substrate Kit can be used in Western immunoblotting techniques as well as in dot blotting assays. The kit includes the 10X solution of CN/DAB and accompanying 1X Stable Peroxide Substrate Buffer.

Features of the CN/DAB Substrate Kit:

HRP substrate—for detection of horseradish peroxidase activity on solid media via the combined action of CN (4-chloro-1-naphthol) and DAB (3,3'-diaminobenzidine tetrahydrochloride)
Chromogenic—no special equipment needed for visualization; yields intense black bands that are easy to photograph
Easy-to-use kit—substrate stock solution and stable peroxide substrate buffer mean that there's no dry powders to measure and dissolve
Stable—store refrigerated at 4°C for at least one year

• CN/DAB Solution (10X) and Stable Peroxide Substrate Solution (1X)

Pierce™ DAB Substrate Kit Thermo Scientific™

The Thermo Scientific Pierce DAB Substrate Kit enables chromogenic detection of horseradish peroxidase (HRP) activity based the action of 3,3'-diaminobenzidine (DAB) in Western blot and tissue staining methods.

DAB (3,3'-diaminobenzidine tetrahydrochloride; MW = 214.1) reacts with HRP in the presence of peroxide to yield an insoluble brown-colored product at locations where peroxidase-conjugated antibodies are bound to samples. The brown precipitate is insoluble in alcohol and other organic solvents, making it an excellent substrate for immunohistochemical staining that requires the use of traditional counterstains and mounting media. The Pierce DAB Substrate Kit includes a 10X solution of DAB and Stable Peroxide Substrate Buffer. Crystalline DAB (powder) is also available.

Features DAB Substrate:

HRP substrate—for detection of horseradish peroxidase activity on solid media, including nitrocellulose and PVDF membranes and fixed tissue samples
Chromogenic—no special equipment needed for visualization; the DAB reaction produces brown bands or spots at sites of reaction with HRP-conjugated antibodies or probes
Package options—choose powder for custom formulation with buffer and peroxide or a complete kit of preformulated solutions for immediate use

Features of the Pierce DAB Substrate Kit:

Easy-to-use—dilute 10X stock solution to 1X in provided stable peroxide buffer
Safer—preformulated DAB solution minimizes safety concerns with this chemical
Complete—includes convenient solution of stable hydrogen peroxide buffer (Part No. 34062)
Stable—store refrigerated at 4°C for at least one year; freezer storage is not necessary

DAB has been used in a variety of applications, including Western blotting, immunohistochemistry and electron microscopy. Many enhancement methods have been reported for DAB involving different buffer conditions, the addition of metal ions and post-treatment applications (Ref. 1-7; also see the Pierce Metal Enhanced DAB Substrate Kit, Part No. 34065). One disadvantage of DAB compared to other substrates is the need to use the working solution immediately because it begins a reaction process upon the addition of hydrogen peroxide. This results in increased background color of the substrate. However, the brown bands resulting from DAB reactions do not fade as quickly as the color-products of other precipitating HRP substrates.

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DAB Substrate Powder (3,3'-diaminobenzidine tetrahydrochloride)

Metal Enhanced DAB Substrate Kit Thermo Scientific™

Thermo Scientific Pierce Metal-Enhanced DAB Substrate Kit uses cobalt and nickel chloride in a special formulation of diaminobenzidine peroxidase substrate that yields intense color for immunohistochemical staining.

Features of the Metal-Enhanced DAB Substrate Kit :

Incredible sensitivity—fifty times more sensitive than the traditional DAB method and thiry times more sensitive than other metal-intensified versions of DAB
Low background, high intensity—get a crisp dark brown-black precipitate that is more intense than the dull brown precipitate when using DAB without enhancement. And, even with the increased intensity, background is almost nonexistent
Only two components—simply mix the two liquid components and your working solution is ready to use
Six-hour stability—the innovative working solution is stable for more than six hours at room temperature, while other DAB substrates must be used immediately

To prepare the product, the 10X Metal Enhanced Substrate Solution is diluted with the Pierce Stable Peroxide Buffer prior to use, and the special formulation allows the 1X substrate to be stable for at least 6 hours at room temperature. The background color of the substrate does not increase and, unlike typical metal enhanced DAB methods, the reactivity is unchanged (1,2). The resulting precipate is a dark brown-black that is more intense than the brown color from DAB alone. The intensity not only increases, but the background is also reduced, which increases the signal-to-noise ratio and makes screening tissue or membrane antigens easier.

Stable DAB Invitrogen™

Stable DAB is a stable, pre-diluted chromogen system designed to clearly localize tissue, DNA, mRNA, and antigens in in situ hybridization and immunoperoxidase procedures. Stable DAB is a ready-to-use reagent containing the correct amounts of diaminobenzidine and hydrogen peroxide. Stable DAB does not fade, creating a permanent record of results under a standard coverslip. It is color coded red for easy user identification.

Diaminobenzidine (DAB) Histochemistry Kit #3, with Streptavidin—HRP Invitrogen™

The DAB Histochemistry Kit for biotinylated probes contains sufficient materials to stain approximately 200 slides using horseradish peroxidase (HRP) and the HRP substrate DAB. The brown-colored DAB reaction product can be visualized by bright-fieldd light microscopy or, following osmication, by electron microscopy.

eBioscience™ DAB Advanced Chromogenic Kit Invitrogen™

This DAB Advanced Chromogenic Kit contains the necessary reagents to prepare a DAB substrate working solution. The enzyme peroxidase (Horseradish Peroxidase/HRP) utilizes the DAB substrate (3, 3’-diaminobenzidine tetrahydrochloride) producing a brownish-red precipitate. These precipiates are insoluble in organic solvent. This enzyme/substrate reaction can be used on tissue sections or cells (grown on culture slides or cytospins) to visualize the localization of an HRP-conjugated antibody in immunocytochemistry and immunohistochemistry applications.

Reported Application
Microscopy, Immunohistochemical Staining, Western Blotting, Immunocytochemistry

Histomount Mounting Solution

This mounting solution is designed for permanent mounting of DAB and BCIP/NBT. Dehydrate tissue before use. This is the same solution that is provided in the Histostain® -SP DAB Kit.

Pierce™ Stable Peroxide Substrate Buffer (10X) Thermo Scientific™

Thermo Scientific Pierce Stable Peroxide Substrate Buffer (10X) include optimized, reliable and convenient stock solutions of peroxide for HRP-based assays detection.

Features of Stable Peroxide Substrate Buffer (10X):

• Ready-to-use
• Minimize assay-to-assay variability

Buffered stable hydrogen peroxide (H2O2) is used by researchers who prefer to make their own HRP-ELISA substrates. This is easily done by adding a chromogen of choice to the buffer. Stable Peroxide Substrate Buffer has a long shelf life (12 months after receipt) and is provided as a 10-fold concentrate. A total volume of 1000 mL can be made from one 100 mL bottle of concentrate. If you use 10 mL of substrate per plate, this will allow you to make substrate for 100 plates from only one bottle of Stable Peroxide Substrate Buffer.

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