I ml. 

 2gm. 



MEDICAL AND BIOLOGICAL STAINING TECHNIQUES 



References : 



* Brachet, J. (1942), Archives de Biol., 53, 207-57. " Localisation des acides 

 pentosenucleiques dans les tissues animaux et les oeufs d'Amphibiens en voie 

 de developpement. " 



Brachet, J. (1953), Q. jf. Micr. Sc, 94, i-io. " The use of basic dyes and 

 ribonuclease for the cytochemical detection of ribonucleic acid. " 



NILE BLUE SULPHATE - SAFRANIN 



An histochemical technique for demonstrating phospho- 

 lipids in frozen sections 



Solutions required: 



A. Formalin 10% . . . . . . 100 ml. 



Calcium Chloride 1% 

 Calcium Carbonate . . 

 Shake well: filter before use. 



B. Safranin 1% aqueous . . . . 100 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. 



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 for ninety 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. 



340 



