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DAB Substrate Kit, Peroxidase (HRP), with Nickel, (3,3'-diaminobenzidine) (SK-4100)



This kit contains all of the reagents necessary to prepare either a DAB or a DAB/nickel substrate working solution. Vector DAB Substrate (3,3’-diaminobenzidine) produces a brown reaction product in the presence of peroxidase (HRP) enzyme. This kit also includes nickel chloride, which, when added to the substrate working solution, results in a gray-black reaction product. Vector DAB Substrate is suitable for darkfield and electron microscopy (EM).

Features of this kit:

  • Ideal for IHC/ICC/ISH and blots
  • Heat Stable
  • Permanent mounting
  • Ideal for single and multiple labeling
  • No wait times for mixing and dissolving powders or tablets
  • Sufficient reagents to produce 300 ml of working solution


Unit Size1 kit
ApplicationsImmunohistochemistry / Immunocytochemistry, In situ hybridization, Blotting Applications, Elispot
Detection Enzyme(s)Peroxidase
Substrate Reaction ProductPrecipitating Reaction Product
Chromogen ColorBrown, Gray-Black
MicroscopyBrightfield, Darkfield, Electron, Spectral Imaging
Additional Substrate PropertiesHeat Resistant, Multiple Labeling
MountingAqueous (Hardening), Non-Aqueous (Permanent)
Safety TitleWARNING: Cancer –

What’s in the box:

  • 6 ml DAB Reagent 1
  • 6 ml DAB Reagent 2
  • 6 ml DAB Reagent 3
  • 6 ml DAB Reagent 4 (Nickel Optional)

Product FAQs

Make a fresh working solution of DAB substrate per instructions. Place a small volume (~1 mL) of this DAB substrate into a clean glass test tube. To this 1 ml aliquot, add one drop (~50 µL) of Reagent B only from the VECTASTAIN® Elite ABC kit. If the HRP enzyme is active and the DAB reacts with it, an immediate color change will be observed. This indicates the end detection reagents are working appropriately.


Technical Information

This kit contains stock solutions in convenient dropper bottles. Slides developed with DAB/ Ni should be dehydrated, cleared, and permanently mounted. For additional information, view this product review on


Counterstain / Substrate Compatibility

This table is designed as a reference to determine the optimal counterstain / substrate combination for your application. Consideration should be given to tissue type, antigen unmasking protocol, and other detection parameters to achieve the desired staining intensity.


SubstrateCatalog NumberHematoxylin (H-3404)
Hematoxylin QS (H-3401)
Methyl Green (H-3402)Nuclear Fast Red (H-3404)
ImmPACT DAB (brown)SK-4105Excellent ContrastExcellent ContrastFair Contrast
ImmPACT DAB EqV (brown)SK-4103Excellent ContrastExcellent ContrastFair Contrast
DAB (brown)SK-4100Excellent ContrastExcellent ContrastFair Contrast
DAB-Ni (gray-black)SK-4100Excellent ContrastFair Contrast*Good Contrast
ImmPACT AEC (red)SK-4205Excellent ContrastCounterstain Incompatibility**Color Incompatibility
ImmPACT AMEC Red (red)SK-4285Excellent ContrastCounterstain Incompatibility**Color Incompatibility
AEC (red)SK-4200Excellent ContrastCounterstain Incompatibility**Color Incompatibility
TMB (blue)SK-4400Color IncompatibilityCounterstain IncompatibilityExcellent Contrast
ImmPACT VIP (purple)SK-4605Fair ContrastExcellent ContrastPoor Contrast
Vector VIP (purple)SK-4600Fair ContrastExcellent ContrastPoor Contrast
ImmPACT SG (blue-gray)SK-4705Poor ContrastGood ContrastExcellent Contrast
SG (blue-gray)SK-4700Poor ContrastGood ContrastExcellent Contrast
ImmPACT NovaRED (red)SK-4805Excellent ContrastExcellent Contrast***Color Incompatibility
Vector NovaRED (red)SK-4800Excellent ContrastExcellent Contrast***Color Incompatibility
ImmPACT Vector Red (magenta)SK-5105Excellent ContrastExcellent ContrastColor Incompatibility
Vector Red (magenta)SK-5100Excellent ContrastExcellent ContrastColor Incompatibility
Vector Black (black)SK-5200Excellent ContrastExcellent Contrast*Excellent Contrast
Vector Blue (blue)SK-5300Color IncompatibilityGood ContrastExcellent Contrast
BCIP / NBT (indigo)SK-5400Color IncompatibilityExcellent Contrast*Excellent Contrast
*This substrate shows a slight decrease in sensitivity following the methyl green protocol. This decrease can be minimized by reducing the heat incubation and acetone rinse times.
**Substrate dissolves in acetone wash.
***A slight color change in ImmPACT NovaRED and Vector NovaRED reaction produt may be seen using methyl green.

SUBSTRATEsensitivity2 jpg

SubstrateCombos jpg

Applicable patents and legal notices are available at legal notices.

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