186 Microscopic Histochemistry 



of activity appear in shades listed in Table 1 (p. 171); back- 

 ground, yellowish. 



The only possible source of error is the staining of entero- 

 chromaffin cells in a reddish shade, but only after formalin 

 fixation. 



The azo dye method (one or the red color variants), fol- 

 lowed by Kossa's technique for Ca, is very suitable for the 

 simultaneous demonstration of preformed calcifications and 

 of sites of phosphatase activity. It should supersede the 

 older cobalt-suLfide-lead-acridine-red method.^^ 



5- Nucleotidase 



The existence of the enzyme 5-nucleotidase has, been re- 

 ported by Reis^^ and by GuUand and Jackson,^* on the basis 

 of chemical studies. The substrates of the enzyme are 5- 

 nucleotides (muscle adenylic acid, inosinic acid, and, possi- 

 bly, adenosinetriphosphoric acid). The pH optimum is 

 around 7.8, but the enzyme is quite active even at pH 9. 



The histochemical method^^ for 5-nucleotidase is very sim- 

 ilar to the method for alkaline phosphatase; in fact, it may 

 be identical with it except for the substrate. However, if 

 sharp differentiation from alkahne phosphatase is desired, it 

 is better to perform incubation at pH ± 8.3. At this pH, 5- 

 nucleotidase is fully active, while the activity of alkaline 

 phosphatase is only about one-third of the maximum. A 

 slight disadvantage of this low pH is a tendency toward 

 diffusion artifacts. It can be offset almost completely by a 

 sufficiently high concentration of Ca ions. 



The substrate used is muscle adenylic acid^^ (regular 



52. Gomori, G.: Am. J. Path., 19:197, 1943. 



53. Reis, J.: Bull. Soc. chim. biol., 16:385, 1934; Reis, J.: Enzymologia, 

 2:110, 183, 1937-38; Reis, J.: ibid., 5:251, 1938-39; Reis, J.: ibid., 2:183, 

 1937-38; Reis, J.: Bull. Soc. chim. biol., 22:30, 1940; Reis, J.: Biochem. J., 

 46:xxi, 1950; Reis, J.: ibid., 48:548, 1951. 



54. Gulland, J. M., and Jackson, E. M.: Biochem. J., 32:597, 1938. 



55. Gomori, G.: Proc. Soc. Exper. Biol. & Med., 72:449, 1949. 



56. Available from the Schwarz Laboratories, 202 E. 44th St., New York 

 17; from Nutritional Biochemicals, Cleveland 28, and from the Sigma Chemi- 

 cal Co., 4648 Easton Ave., St. Louis 13. 



