Histochemical Staining of Alkaline phosphatase, Acid phosphatase and Succinate dehydrogenase

Principles and Methods of Histochemical Staining of Alkaline phosphatase, Acid phosphatase and Succinate dehydrogenase.
Alkaline phosphatase is a generic name for phosphomonoesterases that hydrolyze orthophosphate at an alkaline pH. These enzymes are widely distributed, usually activated by magnesium, manganese, zinc, and cobalt ions and inhibited by cysteine, cyanides, and arsenates. This is a simultaneous coupling azo dye method first developed in 1944. It has been modified repeatedly. Sodium α-naphthyl acid phosphate, the substrate used, is hydrolyzed and then coupled to the diazonium salt (Fast Blue RR) which is then precipitated at the site of enzyme activity.
The complex naphthol, naphthol acid phosphate, is hydrolyzed by acid phosphatases present in the tissue, and naphthol derivatives are therby produced. The naphthol derivatives couple with the unstable diazonium salt, hexazonium pararosanilin, and other dyes (rosaniline, magenta II & new fuchsine) to produce a red azo dye that marks the site of enzyme activity.
Succinic dehydrogenase (SDH), is a soluble iron flavoprotein that catalyzes the reversible oxidation of succinic acid to fumaric acid. The histochemical demonstration of the activity of this enzyme is achieved by incubation of fresh frozen sections with a succinate substrate in the presence of a tetrazolium compound. Tetrazoliums are water-soluble compounds employed in histochemistry as redox indicators. Under appropriate conditions, tetrazoliums are reduced to formazans which are water-insoluble tetrazolium (NBT). Enzymatic activity releases hydrogen from colored compounds. Commonly used tetrazoliums include nitro blue the substrate, and the released hydrogen is transferred to the tetrazolium. With the addition of hydrogen, the tetrazolium is converted to purple-blue formazan pigment marking the site of enzyme activity.
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