Redox Systems for Electrochemical Detection of Alkaline Phosphatase, Horseradish Peroxidase, β-Galactosidase, Glucose Oxidase, Choline Oxidase, and Acetylcholine Esterase

Two new substrate chromogens for alkaline phosphatase (ALP) detection have been recently synthesized for use in molecular biology research, salmon and magenta phosphate. We show here that these two chromogens have advantageous characteristics for immunocytochemistry as well. Their relatively delicate pink- and magenta-colored products do not mask the colors produced by other staining procedures. In addition, the reaction products of these substrates are insoluble in water, ethanol, and xylene, permitting the use of regressive hematoxylin staining procedures and coverslipping in permanent resin-based media. Most importantly, when these ALP substrates are used in double-label immunocytochemistry in combination with horseradish peroxidase-diaminobenzidine (HRP-DAB) and counterstained with hematoxylin, all three colors can be easily distinguished. An application using these substrates for simultaneous immunocytochemical detection of two monoclonal antibodies of different classes, in combination with hematoxylin staining, is illustrated.

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One-step double immunolabeling of mouse interdigitating reticular cells: simultaneous application of pre-formed complexes of monoclonal rat antibody M1-8 with horseradish peroxidase-linked anti-rat immunoglobulins and of monoclonal mouse anti-Ia antibody with alkaline phosphatase-coupled anti-mouse immunoglobulins.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.12.1940324 ◽

1991 ◽

Vol 39 (12) ◽

pp. 1719-1723 ◽

Cited By ~ 7

Author(s):

T Krenács ◽

H Uda ◽

S Tanaka

Keyword(s):

Alkaline Phosphatase ◽

Horseradish Peroxidase ◽

B Lymphocytes ◽

Enzyme Reaction ◽

Molecular Complexes ◽

Simultaneous Application ◽

One Step ◽

Reticular Cells ◽

Secondary Antibodies ◽

Double Immunolabeling

A novel one-step double immunolabeling method was elaborated on the basis of the simultaneous application of preformed molecular complexes of two primary antibodies with their specific secondary antibodies labeled with different enzymes. Treatment with a rat monoclonal antibody (MAb), M1-8, pre-coupled with horseradish peroxidase-linked sheep anti-rat immunoglobulins, and enzyme reaction revealed by the 3-amino-9-ethylcarbazole/hydrogen peroxide reaction, resulted in red-brown intracytoplasmic staining of interdigitating reticular cells in the lymph nodes of Balb/c mice. Another molecular complex, made of mouse anti-Ia MAb with alkaline phosphatase-linked rabbit anti-mouse immunoglobulins, applied at the same time and then developed with naphthol AS-BI-phosphate/fast blue BB as substrate, yielded blue surface staining of this cell type in addition to labeling of B-lymphocytes. The method described provides the possibility of relatively rapid double antigen detection where the binding sites of the secondary antibodies are saturated by the specific primary immunoglobulins. This approach seems to avoid nonspecific binding of primary antibodies to Fc receptors, and the unwanted binding of secondary antibodies with cell surface immunoglobulins on B-lymphocytes or with crossreactive primary antibodies used in the other sequence, if the primary antibodies and the tissue are the same or crossreactive animal species.

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