242 Pigments and Minerals (chap. 17) 



muscle, myelin, erythrocytes — unstained 

 nuclei — greenish to unstained 



comments: 



Lipofuscins stain with Nile blue by two mechanisms: a fat solubility 

 method operating at pH below 1.0; and an acid-base mechanism oper- 

 ating at levels above pH 3.0. When stained by the second method, 

 they retain a green stain after acetone or brief alcoholic extraction, 

 but when the first mechanism is used, they are promptly decolorized 

 by acetone or alcohol. {Lillie, 1936 B) 



Melanins stain with basic dyes at pH levels below 1.0 and retain 

 the stain when dehydrated and moiuited. [Lillie, 1955) 



Ferric-Ferricyanide Method (lillie, 1957) 



fixation: avoid chroma te fixatives, others are satisfactory. 



solutions: 



Ferrous sulfate: 



ferrous sulfate (FeS04-7H20) 2.5 gm. 



distilled water 100.0 ml. 



Potassium ferricyanide: 



potassium ferricyanide (K3Fe(CN)6) 1.0 gm. 



distilled water 99.0 ml. 



glacial acetic acid 1.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water, 



2. Treat with ferrous sulfate: 1 hour. 



3. Wash in distilled water, 4 changes: total 20 minutes. 



4. Treat with potassium ferricyanide: 30 minutes. 



5. Wash in 1% acetic acid (1 ml./99 ml. water): 1-2 minutes. 



6. Counterstain if desired, picro-ponceau satisfactory, do not use 

 hematoxylin. 



7. Dehydrate, clear, and moimt. 



results: 



melanin — dark green 



backgroiuid — faint greenish or unstained; with picro-ponceau, col- 

 lagen stains red and muscle and cytoplasm will be yellow and brown 



comments: 



Lillie says this method is highly selective. No other pigments react 

 in this procedure, except occasionally hemosiderin. 



