Postcoupling, Noncoupling, and Fluorescence Techniques for the Demonstration of Alkaline Phosphatase

Abstract

The development and use of new post-coupling, noncoupling, and fluorescence techniques for the demonstration of alkaline phosphatase are described. The techniques utilize new substrates, derivatives of hydroxynaphthoic acid, and complex amines. Three of the most useful substrates are 5,6,7,8-β-tetralol carboxylic acid-β-naphthylamide phosphate (I), 2-hydroxy-3-naphthoic acid-2-aminoanthraquinonylamide phosphate (II), and 2-hydroxy-3-naphthoic acid-aminoazobenzanilide phosphate (III). The techniques, which are designed primarily for use with paraffin-embedded tissues, give sharp localizations. These compare with the best results obtained by the simultaneous-coupling technique. In the case of one substrate, an anthraquinone derivative (II), postfixed frozen sections give useful results. The naphthol released from substrate I is a fluorochrome and is the basis of a new avenue of approach in enzyme histochemistry, namely, the visualization of enzyme activity by fluorescence. This and another substrate also liberate naphthols, which are visible under optical magnification without postcoupling. This application is referred to as a noncoupling procedure. For most purposes, a postcoupling procedure utilizing substrates II or III is recommended. The incubating solutions are stable, and subsequent postcoupling may be accomplished with a large variety of diazonium salts. These techniques also lend themselves to the demonstration of hydrolytic enzymes other than alkaline phosphatase.