LIPIDS AND CHOLESTEROL 39 



exclude the presence of cholesterol or its esters. The Windaus 

 digitonin test for free cholesterol (Lison, 1936, pages 211-212) 

 requires further investigation in the opinion of Kay and Whitehead 

 in Lee's Vade Mecum (1937, page 281). By means of the polarizing 

 microscope, cholesterol crystals can occasionally be observed in 

 sections as birefringent rhombic plates. If the temperature is low- 

 enough, neutral fats and fatty acids can also be observed in some 

 instances as birefringent crystals. 



Kay and Whitehead Procedure for Sudan IV 

 Stain for Lipids 



SPECIAL REAGENTS 



Stock Solution of Dye (can be used for at least 6 months). Prepare 

 a saturated alcoholic solution by boiling 2 g. dye in 1 1. absolute 

 alcohol; allow to cool. 



Staining Solution (good for only about 4 hr. after being mixed). 

 Add slowly, with stirring, to 7 vol. stock soln., 9 vol. of 45% 

 alcohol. Filter after standing for 1 hr. The 45% alcohol is prepared 

 by mixing 4 vol. absolute alcohol with 5 vol. distilled water. 



PROCEDURE 



1. Place formalin-fixed frozen sections in 50% alcohol for 5 min. 

 in staining soln. for 30 min. at 37° (turn sections over after 15 min. 

 for more even staining), in 50% alcohol several sec, and finally in 

 distilled water a few min. 



2. Pass through filtered hemalum and wash in alkaline tap water 

 for several min. 



3. Mount in glycerin jelly. 



Result. The lipid will be stained red. -^^ 



The sections should be stained the day after cutting since they 

 tend to be sticky for a while just after the cutting. On the other 

 hand, poor results are often encountered if the staining is delayed 

 longer than one day after sectioning due, presumably, to crystalli- 

 zation of lipid material. The stain lasts for only a few months. 



Jackson Procedure for Lipids Using Acetic-Carbol-Sudan III 



SPECIAL REAGENTS 



Sudan III Stock Solution. Cover 2 g. of the finely pulverized dye 



