Detecting Horseradish Peroxidase-Labeled Cells

Abstract

A range of substrates is available for detection of cells labeled with horseradish peroxidase (HRP). Diaminobenzidine (DAB) is the most commonly used substrate and one of the most sensitive. It yields an intense brown product that is insoluble in both water and alcohol. DAB staining is compatible with a wide range of common histological stains. DAB can be made more sensitive by adding metal salts such as cobalt or nickel to the substrate solution. The reaction product is slate gray to black, and the products are stable in both water and alcohol. DAB/metal staining is compatible with a wide range of common histological stains. Alternatively, chloronaphthol gives a blue-black product and aminoethylcarbazole (AEC) yields a red product. These substrates are less sensitive than DAB and the products are soluble in alcohol, but they can be used if the DAB reaction gives too high a background or if alternative product colors are required. Diaminobenzidine (DAB) tetrahydrochloride (see Steps 1-6 and 7-13) Nickel chloride or cobalt chloride (0.3%, w/v) in H 2 O (see Steps 7-13) Tris buffer (0.05 M, pH 7.6) (for detection using DAB or chloronaphthol) Equipment Whatman No. 1 filter paper From the Antibodies collection, edited by Edward A. Greenfield.

Application of Au@Pt Nanozyme as Enhancing Label for the Sensitive Lateral Flow Immunoassay of Okadaic Acid

“…Nine compositions of the DAB substrate were tested ( Table S1 , solutions 2–9). All of them contained DAB and hydrogen peroxide at different ratios and, in some cases, cobalt or nickel chlorides, which are usually added to the DAB substrate to enhance the coloration of the product of the catalytic reaction [ 36 ]. All substrates were converted to insoluble brown-colored precipitates.…”

Binding Antibodies to Attached Cells or Tissues in Preparation for Staining

“…If the primary antibody is labeled, proceed to detection (see, e.g., Protocol: Detecting Horseradish Peroxidase-Labeled Cells [Rodig 2019a], Protocol: Detecting Alkaline Phosphatase-Labeled Cells [Rodig 2019b], Protocol: Detecting β-Galactosidase-Labeled Cells [Rodig 2020a], Protocol: Detecting Gold-Labeled Cells [Rodig 2019c] or Protocol: Detecting Iodine-Labeled Cells [Rodig 2019d]). If the primary antibody is unlabeled, continue with Step 6.…”