r 



GLYCOGEN AND MUCIN 49 



formol solution for 24 hr. (Deane, Nesbett, and Hastings, 1946, 

 recommend the use of ice-cold alcohol-picric acid-formalin to pre- 

 serve the glycogen throughout the tissue block.) Wash in absolute 

 alcohol and embed in paraffin, being careful to prevent overheating. 

 (It is essential that very fresh tissue be used since glycogen is 

 rapidly autoiyzed. The Altmann-Gersh freezing and drying tech- 

 nique for fixation may also be used; in fact it can lead to a truer 

 picture of the glycogen distribution, as shown by Bensley and 

 Gersh, 1933a) . 



2. Section, mount on slides, and deparaffinize as usual. 



3. Place in 4% chromic acid 1 hr. or in the 1% soln. overnight. 



4. Wash in running water for 5 min., place in Feulgen reagent 

 10-15 min., rinse with three changes of bisulfite soln. for 1.5 min. in 

 each change, and wash in running water for 10 min. 



5. Nuclei may be counterstained with hematoxylin. 



6. Dehydrate, clear, and mount in balsam. 



7. As a negative control, remove the glycogen from some of the 

 sections, brought down to water, by adding fresh saliva. During a 

 15-30 min. period, change the saliva several times. Wash with water 

 at 37° to remove mucus and stain as above beginning with step 3. 

 Comparison of these sections with those not given the saliva treat- 

 ment helps to distinguish the glycogen regions. 



Result. The glycogen appears deep red-violet, the nuclei laven- 

 der. 



Best Carmine Stain for Glycogen (after Bensley) 



SPECIAL REAGENTS 



Alcohol-Formalin Fixative. Same as the reagent for Bauer-Feulgen 

 stain. 



Carmine Stain Stock Solution. Gently boil 2 g. carmine, 1 g. potas- 

 sium carbonate, and 5 g. potassium chloride in 60 ml. distilled 

 water until color darkens. After cooling, add 20 ml. cone, am- 

 monia and let stand 24 hr. This solution may deteriorate in a 

 month in a warm room ; keep well stoppered. 



Fresh Carmine Stain. 10 ml. stock soln., 15 ml. cone, ammonia, and 

 30 ml. methanol (C.P.). 



