152 Microscopic Histochemistry 



much higher pH values all the reagents mentioned will un- 

 dergo nonenzymatic reduction by a number of substances, 

 such as sulfhydryl compounds, polyphenols ( adrenalin ) , etc. 



Method 



According to Seligman and Rutenburg^^ the tissue need 

 not be absolutely fresh; refrigeration for 4 hours at 4° C. 

 does not cause any noticeable loss of activity; in fact, even 

 fixation in chilled acetone for 4 hours causes only 40 per 

 cent inactivation of the enzyme. 



Use frozen sections 25-50 ^ thick. Thinner sections often 

 fail to stain ( destruction of cellular organization in the super- 

 ficial layers or loss of cof actors by diffusion). Zweifach, 

 Black, and Shorr^^ recommend the use of razor-blade hand 

 sections, about 0.5-1 mm. thick. After the reaction has taken 

 place, the pieces can be fixed in formalin, frozen, and cut. 

 Loose cellular material (sediments, scrapings) are sus- 

 pended in the incubating medium. 



The composition of the substrate mixture is not critical, 

 except for the pH ( 7.3-7.6 ) . The concentration of the buffer 

 (phosphate) should be 0.05-0.1 M; that of the substrate (suc- 

 cinate, lactate, etc.), 0.1-0.2 M. Triphenyltetrazolium chlo- 

 ride and neotetrazolium chloride^^ (a dimer of the former) 

 are used in 0.5-1 per cent solutions; the more insoluble ditet- 

 razolium chloride^^ (a dimethoxy derivative of neotetrazo- 

 lium ) and potassium tellurite ( K2Te03 ) in a 0.1 per cent solu- 

 tion. Incubation time at 37° C. will range from 20 minutes to 

 3 hours. The dye formed from triphenyltetrazolium is scarlet- 

 red; that from neotetrazolium, purple-red; that from ditetra- 

 zolium, in thin layers purple, in thick layers deep blue. The 

 shdes should be examined within a few hours because for- 



25. Seligman, A. M., and Rutenberg, A. M.: Science, 113:317, 1951. 



26. Zweifach, B. W., Black, M. M., and Shorr, E.: Proc. Soc. Exper. Biol. 

 & Med., 74:848, 1950. 



27. Available from Pannone Chemical Co., Farmington, Conn. 



28. Available from Dajac Laboratories, 511 Lancaster Ave., Leominster, 

 Mass. 



