Alkaline phosphatase histochemistry

Kiyama, H., Emson, P.C., and Tokyama, M. (1992) In situ hybridization histochemistry using alkaline phosphatase-labeled oligodeoxynucleotide probe. In Methods in Molecular Biology, Protocols in Molecular Neurobiology, (A. Longstaff, and R Revest, eds.), Vol. 13, pp. 167-179. Humana Press, Totowa, New Jersey. 

Goreau, T. F. Histochemistry of mucopolysaccharide-like substances and alkaline phosphatase in Madreporaria. Nature 177, 1029 (1956)... 

Enzyme substrates- A variety of different substrates for both alkaline phosphatase and peroxidase are available. In this chapter, only the alkaline phosphatase substrate nitroblue tetrazolium/bromochloroindolylphosphatase (NBT/BCIP) and the peroxidase substrate 3-amino-9-ethyl carbazole (AEC) are described. Other substrates can be found in textbooks of histochemistry (see also Chapter 24) a NBT/BCIP is made in advance, and stored at -20°C. After equilibration of 30 mL of alkaline phosphatase substrate buffer at 37°C, 10 mg of NBT are dissolved m 200 pL of dimethylformamide (DMF), and added to I mL of the prewarmed substrate buffer. The mixture is added dropwise to the remaining substrate buffer BCIP (5 mg), dissolved in 200 pL of DMF, is then added slowly, and the whole preparation stored m 4-mL aliquots at—20°C b AEC is prepared fresh daily by dissolving 2 mg of AEC in 1 2 mL of dimethyl-sulfoxide in a glass tube. The mixture is added to 10 mL of 20 mM acetate buffer, pH 5 0-5.2. Immediately prior to use, 1 pL of 30% (v/v) hydrogen peroxide is added The final mix may require filtration prior to use... 

Dixit, P. K. Quantitative histochemistry of cartilage. Alkaline phosphatase and glucose-6-phosphate dehydrogenase activity in different zones of rachitic rat cartilage during healing. Calc. Tiss. Res. JO, 49-57 (1972). 

Enzyme substrates. A variety of different substrates for both alkaline phosphatase and peroxidase are available. In this chapter, only the alkaline phosphatase substrate nitroblue tetrazolium/bromochloroindolylphos-phatase (NBT/BCIP) and the peroxidase substrate 3-amino-9-ethyl car-bazole (AEG) are described. Other substrates can be found in textbooks of histochemistry (see a o Chapter 10). 

The location of alkaline phosphatase in the cell and tissue is information basic to a full explanation of an increase in serum alkaline phosphatase. Modern techniques of enzyme histochemistry and of electron microscopy (R2) have succeeded in providing a clearer picture (GIO, M22). The cell wall is the location at the electron microscope level of... 

Serum isozymes following different types of injuries. The multimolecular forms of enzymes, called isozymes, may be identified using combinations of suitable gel electrophoresis and histochemistry. The changes in naphthylamidase and alkaline phosphatase following burns most likely indicate an alteration of liver metabolism and may persist for several weeks (A5). A rise in plasma alkaline phosphatase, but of kidney origin, can be found after limb ischemia in the rabbit. 

Visualizing lacZ by Immunostaining or Histochemistry in Combination with Alkaline Phosphatase Histochemistry... 

A more critical approach to histochemical problems was heralded by the publication of Lison s Histochemie Animale in 1936 (55). Some of Lison s original studies dealt with the meaning of metachromasia, the effects of fixatives on glycogen, and other topics of carbohydrate histochemistry. Other milestones in histochemistry were the alkaline phosphatase method of Gomori (57) and the periodic acid - Schiff method for carbohydrates developed by McManus (55), Lillie 59), and Hotchkiss (50). The technical performance and fruitful results of these two methods did much to show the possibilities of histochemistry in histology and pathology. 

There are many commercially available labeled secondary antibody products. The most common conjugated enzymes are alkaline phosphatase and horseradish peroxidase. Alkaline phosphatase, discussed earlier in the section on histochemistry, dephosphorylates the substrate 5-bromo-4-chloro-3-indolyl phosphate which can be oxidized by nitroblue tetrazolium. The resulting signal would appear as a blue/ purple dye. Horseradish peroxidase can catalyze the oxidation of 4-(chloro-l-naphthol), in the presence of hydrogen peroxide, into an insoluble blue dye. Apart from using antibody-enzyme conjugates, secondary antibodies can be labeled with to permit detection by autoradiography. 

---