DAB Chromogenic Substrate Kit (Blue)

List of Components

Overview

Assay Principle

DAB (3,3′-diaminodbenzidine) is a sensitive colorimetric substrate used with horseradish peroxidase (HRP) in immunohistochemistry and immunoblotting applications. The DAB Chromogenic Substrate Kit (Blue) contains the necessary reagents to prepare a DAB substrate working solution. The DAB working solution reacts with peroxidase (Horseradish Peroxidase/HRP) detection system to yield an insoluble Blue precipitate. This diaminodbenzidine-based peroxidase substrate can be used on tissue sections and cells to visualize the localization of an HRP-conjugated antibody in immunocytochemistry and immunohistochemistry applications.

Important Product Information

DAB is a suspected carcinogen. Good laboratory practices should be followed. Avoid contact with skin and eyes and use appropriate protective equipment when handling DAB, including gloves, protective eyeware and a lab coat. Dispose in accordance with local regulations.

Assay Protocol

1. Following incubation with a peroxidase (HRP) detection antibody, wash slides three times with TBS or PBS for 7 minutes each.

2. To prepare the DAB working solution, add one drop(50μL) Reagent A (DAB Chromogen Concentrate), one drop (50μL) Reagent B (H2O2 Concentrate) and one drop(50μL) Reagent C (TBS buffer Concentrate) to 1mL distilled water and mix well.

3. Cover the slide completely with the DAB working solution and incubate at room temperature until desired color intensity develops.1-10minutes generally provides an acceptable staining intensity.
Note: For best result, monitor slides during development process using a microscope. The concentration of DAB working solution could be increased or decreased according to the staining intensity.

4. Wash the slide well with water.

5. Counterstain with nuclear fast red for 5 minutes, if desired.

6. Dehydrate, clean in xylene and mount slide.