Saccharides (Carbohydrates) 271 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Treat with colloidal iron: 10 minutes. 



3. Wash 'well in distilled water. 



4. Treat ^vith potassiinn ferrocyanide: 10 minutes. 



5. Wash well in distilled water. 



6. Flood with 70% alcohol. 



7. Treat wdth periodic acid: 5 minutes. 



8. Rinse ^vith 70% alcohol. 



9. Transfer to reducing: rinse: 5 minutes. 



10. Rinse with 70% alcohol. 



11. Transfer to Schiff reagent: 1 hour. 



12. Use -^ sulfite rinses: 1.5 minutes in each. 



13. Wash well in rinming water: 5 minutes. 



14. Stain with hematoxylin if desired. 



15. Dehydrate, clear, and mount. 



results: 



acid polysaccharides — blue 

 glycogen — magenta 



comments: 



Nito and Stokes (1960) prepare a dialyzed iron as follows: Dissolve 

 100 gm. ferric chloride in 230 ml. distilled water. Add 130 ml. 

 glycerol and mix well. Divide into 2 portions of ^ and f . To the large 

 volume add concentrated ammonia in small quantities with vigorous 

 stirring. A precipitate forms and dissolves more and more slowly 

 until the mixture becomes pasty (approximately 110 ml. ammonia). 

 Add drop by drop and with constant stirring the smaller portion until 

 the solution becomes clear dark reddish brown. It now contains a 

 slight excess of ferric chloride. 



Dialyze against distilled water using a cellophane bag — capacity 

 about 4 times that of the soltuion. Change the distilled water every 

 day initil the soltuion in the bag has reverted to its original \olume 

 (about 2 weeks). Store at room temperature. For use diltite with equal 

 amount of water. 



Acid mucopolysaccharides combine with dialyzed iron and then 

 become blue in potassitun ferrocyanide (Prtissian blue). Mowry 

 (1938) considers this method more specific than others, and it stains 

 more strongly than alcian blue. 



