Melanin and Lipofuscin Staining 241 



comments: 



This reaction can not be considered reliable at all times because the 

 reactants are diffusible; also the final color can spread from the 

 original site. The test is based on oxidation ot the bile pigment 

 (bilirubin) to green biliverdin by the iodine solution. 



Melanin and Lipofuscin Staining 

 Lillie (1956 A, B), Nile Blue Method 

 fixation: any good general fixative. 



SOLUTION : 



Nile blue A: 



Nile blue A, C.I. 51180 0.05 gm. 



sulfuric acid, 1% (1 ml. cone. HoSO^/QQ ml. dis- 

 tilled water) 100.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water; remove HgCU. 



2. Stain in Nile blue A solution: 20 minutes. 



3. Wash in running water: 10-20 minutes. 



4. Mount in glycerol jelly. 



results: 



lipofuscins— dark blue or blue green 



melanin^ — pale gTeen 



erythrocytes — greenish yellow to gTcenish blue 



myelin — green to deep blue 



nuclei — poorly stained 



Alternate Method: 

 procedure: 



Steps 1 and 2 as above. 



3. Do not wash in water. Rinse quickly in 1% sulfuric acid to remove 

 excess dye. 



4. Dehydrate at once, acetone, 4 changes: 15 seconds each. 



5. Clear in xylene and mount. 



results: 



cutaneous, ocular, meningeal melanins — dark green 



mast cells — purple red 



lipofuscins — imstained but appear yellow to brownisli 



