258 Cytoplasmic Elements (chap. 18) 



Nile Blue A (Sulfate) (mallory, 1938) 



This is a useful method for separating neutral fats from other fats. 



fixation: 10% formalin, no alcohol. 



solution: 



Nile blue A: 



Nile blue A, C.I. 51 180 1.5 gm. 



distilled water 100.0 ml. 



procedure: 



1. Frozen sections, 15 microns, into distilled water. 



2. Stain in Nile blue sulfate: 20 miniues. 



3. Rinse in tap water. 



4. Differentiate in 1% acetic acid (1 ml./99 ml. Avater), 10-20 min- 

 utes, or imtil colors are clear; can happen in 1-2 minutes. 



5. Wash thoroughly, distilled water, several changes. 



6. Mount in glycerol jelly or ^6o/;o^^. 



results: 



neutral fats — pink to red 



fatty acids — blue to violet 



nuclei and elastic tissue — dark blue 



comments: 



There appears to be some doubt about the specificity of this reaction 

 - — whether it actually differentiates between neutral fats and fatty 

 acids. Gomori (1932) says that not all structures staining pinks, reds, 

 or blue are lipid. Use the method with tongue in cheek. 



Staining for Phospholipids (Elftman, 1957B) 



fixation: 



mercuric chloride, 5% aqueous 100.0 ml. 



potassium dichromate 2.5 gm. 



Mix fresh. Fix for 3 days; oxidizes phospholipids and makes them in- 

 soluble in fat solvents such as are used in paraffin embedding. 



solutions: 



Sudan black B: 



Dissolve 0.7 gm. Sudan black B, C.I. 26150, in 100.0 ml. ethylene gly- 

 col with heat, 100-1 10°C (not above). Stir. Filter while hot through 

 Whatman #2 filter paper. Cool and filter through fritted glass filter 

 of medium porosity with suction. 



