SECTION TWO 



NILE BLUE SULPHATE - SAFRANIN 



An histochemical technique for demonstrating phospho- 

 lipids in frozen sections 



Solutions required: 



Formalin io% 



loo ml. 



I ml. 

 2gm. 



Calcium Chloride i% 

 Calcium Carbonate . . 

 Shake well : filter before use. 



B. Safranin i% aqueous . . . . loo ml. 

 Aniline Oil . . . . . . . . 2 drops 



C. Nile blue sulphate, saturated aqueous 100 ml. 

 Sulphuric acid 0-5% . . . . 10 ml. 

 Boil for 2 hours under reflux condenser. 

 Filter before use. 



D. Acetic acid 5% aqueous. 



E. HCl cone. . . . . . . . . 0-5 ml. 



Distilled water 99-5 ml. 



Technique: 



1. Fix material in solution A: then cut frozen sections, without 

 embedding: or the material may be embedded, if desired, in 

 gelatine or carbowax or waterwax. 



Note: Frozen sections should not be stored in water for more 

 than ten to fifteen minutes. 



2. Stain in the safranin solution for five minutes, 



3. Rinse in distilled water. 



4. Stain in the nile blue sulphate forfninety minutes at 60^ C. 



5. Rinse in distilled water. 



6. Immerse in acetone heated to 50° C. on a water bath. 



7. Remove the acetone from the source of heat and allow the 

 sections to remain in it for half an hour. 



8. Differentiate in 5% acetic acid for thirty minutes. 



9. Rinse thoroughly in distilled water. 



ID. Refine the differentiation in the 0-5% HCl (Solution D). 

 1 1 . Wash in several changes of distilled water. 

 17. Mount in glycerine jelly or in Aquamount. 



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