.142 Histochemistry (chap. 21) 



2. Place in incubating solution, 37°C: 0.5-3 hours. (Warm soliuion 

 up to 39°C before starting incubation.) 



3. Rinse in distilled water. 



4. Change to colored form by either of following methods: 



A. Immerse in 1-2% cobalt salt (chloride, nitrate, acetate or sul- 

 fate) (1-2 gm./lOO ml. water): 3-5 minutes. 



Rinse thoroughly, distilled w^ater, 3 changes. 



Immerse in fresh dilute solution of yellow ammonium sulfide (5-6 



drops in coplin jar of distilled water). 



Rinse in distilled water, 3-4 changes. 



Cotmterstain if desired, dehydrate, clear, and motnit. 



B. Immerse in 5% silver nitrate (5 gm./lOO ml. water) luider ul- 

 traviolet lamp: 30 minutes. Or use 0.5% silver nitrate (0.5 gm./ 

 100 ml. water) in direct sunlight, depending on light intensity. 

 Rinse in distilled water. 



Fix in 1-2%, sodium thiosulfate: 2 minutes. 

 Rinse, counterstain, dehydrate, clear, and moimt. 



Counterstains may be phloxine-methylene blue, PAS, eosin, safranine, 

 or the like. 



results: 



cobalt method — brownish black 

 silver method — golden brown 



comments: 



False positives can be due to hemosiderin or melanin and these pig- 

 ments shoidd be checked for presence. 



References: Ackerman (1958); Gomori (1941 A, 1946); Kabot and 

 Furth (1941); and Moffat (1958) 



Azo-Coupling Method 



The sections are incubated in a soliuion of sodium alpha naphthyl 

 phosphate and a diazonium salt. The phosphatase activity liberates 

 alpha naphthol to couple with the diazoniiun salt and form an insoluble 

 pigment. 



fixation: cold 10% formalin (4°C): 8-16 hours. 



Froze?! sections, 10-15 microns: mount on clean slides with no ad- 

 hesive, air-dry 1-3 hours. 



