/•>// Staining 253 



Fat Staining 



Lipids (synonyms: lipoids, fatty substances) include a large number 

 of substances grouped together because of their solubility properties. 

 They are insoluble in ^vater and soluble in fat solvents: alcohol, ether, 

 chloroform pyridine, benzene, and acetone, to name a few. Classifying 

 the lipids is not to be undertaken here, but a few familiar groups can 

 be mentioned: carotenoids (vitamin A), fatty acids, triglycerids (neutral 

 fats), phosphatids, and lipid pigments (lipofiiscins — page 232). 



For the fixation of fats, formalin is best, particularly if 1% of calcium 

 chloride is added to make the phospholipids insoluble (Gomori, 1952). 

 Because of the use of fat solvents, the tissue cannot be embedded in 

 paraffin or nitrocellulose, but it can be embedded in carbowax. Frozen 

 sections are simpler and most frequently used. During any processing 

 alcohol higher than 70% must be avoided. (Concerning the fixation of 

 lipoids, see Elf t man, 1958.) 



The dying of fats is one of the simplest forms of staining; the color- 

 ing agent merely dissolves in a fluid contained ^vithin the tissues. In 

 addition, it should be einphasized that a dye solvent should be used 

 which does not dissolve the lipid itself. The dye, therefore, must meet 

 certain requirements: 



1. The dve must be stronfflv colored. 



2. It must be soluble in the substance which it is intended to show, but 

 must not be soluble in water, the major constituent of cells. 



3. It must not attach itself to any tissue constituents except by solution. 



4. It must be applied to tissues in a solvent which will not dissolve the sub- 

 stance to be dyed, and must be less soluble in the solvent than in the 

 substance. 



Baker (1958) suggests the name "Tysochromes" for these dyes that 

 dissolve in the tissue elements to be colored. The name is deri\ ed from 

 the Greek lusis meaning solution. The Sudans were the first synthetic 

 dyes of this sort, followed by the Nile blues and reds. 



These dyes are used in saturated solutions and often introduce a 

 problem of dye precipitate on the tissue. Vlachos (1959) makes a sensi- 

 ble proposal that the precipitate probably is formed by the solution be- 

 ( oming oversaturated and that perhaps a saturated solution is unneces- 

 sary. He suggests two alternatives: 



1. Make up the concentration below the saturation point; for example, 0.25 

 gm. Sudan IV per 100 ml. m% alcohol. 



