STAINING, PRACTICAL AND THEORETICAL 



Results: 

 Phospholipids: blue. Nuclei: red. 



Reference: Menschik, Z. (1953). 



NILE BLUE 



For the differentiation of melanins and lipofuscins 



(After Lillie, 1956) 



Solution required: 

 Nile blue A . . . . . . ot gm. 



Sulphuric acid, cone. . . . . 100 ml. 



Technique: 



1. Fix pieces of tissue in 10% formalin or other appropriate 

 fixative, and embed in paraffin wax. 



2. Mount sections on slides and dewax with xylol. 



3. Pass through the usual graded alcohols to distilled water. 



4. Stain in the Nile blue solution for twenty minutes. 



5. Wash in running water for ten to twenty minutes. 



6. Mount in glycerine jelly or in Aquamount. 



Results: 



Lipofuscins: dark blue or greenish blue. Melanins: dark 

 green. Cytoplasm, muscle: pale green. Erythrocytes: greenish 

 yellow to greenish blue. Myelin: green to deep blue. Nuclei: 

 unstained or only faintly stained. 



Notes: 



The method is based on Professor Lillie's findings that: 



{a) Melanins stain with basic dyes at pH levels below i -o and 

 retain the stain when dehydrated and mounted in resinous media. 



{b) Lipofuscins stain with Nile blue by two mechanisms, a fat 

 solubility one which operates at pH levels below i-o, and an 

 acid-base mechanism operating at pH levels above 3 -o. 



When stained by the second of these mechanisms, lipofuscins 

 retain their green stain after acetone or brief alcoholic extraction; 

 but when the first mechanism is applied they are promptly 

 decolorized. 



Reference: Lillie, R. D. (1956a, 1956&, 1955). 



