Organic Substances 99 



cent cholesterol show no birefringence.^^ Even a positive re- 

 sult is not necessarily evidence of the presence of choles- 

 terol.^^ 



Chemical Methods 



A) For fatty acids and insoluble soaps (Ca).— These sub- 

 stances can be demonstrated by Fischler's method.^" 



Method 



Mordant frozen sections in a half-saturated solution of cu- 

 pric acetate for 3-12 hours around 37° C; rinse sections 

 thoroughly in repeated changes of distilled water and stain 

 them in a 0.5 per cent solution of hematoxylin in 50 per cent 

 alcohol for 12-24 hours. Differentiate in Weigert's borax- 

 ferricyanide mixture until the background (nuclei included) 

 is decolorized. The cupric soaps are very resistant to decol- 

 orization and will remain almost black. 



The reaction is not too specific; red cells, muscle, and cal- 

 careous deposits of any sort are also intensely stained. Two 

 control sections can be used: one from which lipids are re- 

 moved with warm methyl alcohol or chloroform (preferably 

 with about 10 per cent acetic acid added to decompose the 

 soaps) and another one treated with a citrate buffer of 

 pH 4.5-5 to remove other calcium deposits. 



The use of Nile blue has been mentioned. Faure-Fremiet^^ 

 has reported that unsaturated fatty acids stain metachromat- 

 ically with methyl green and hght green; his findings could 

 not be confirmed. 



B) For cholesterol and its esters.— 



a) Schultz's method^* is the histochemical apphcation of 



SO. Okey, R.: J. Biol. Chem., 156:179, 1944. 



31. Yoffey, J. M., and Baxter, J. S.: J. Anat, 81:335, 1947. 



32. Fischler, F.: Zentralbl. f. allg. Path. u. path. Anat., 15:913, 1904. 



33. Faure-Fremiet, Mayer A., and SchaeflFer, G.: Arch, d'anat. micr., 

 12:19, 1910. 



34. Schultz, A.: Zentralbl. f. allg. Path. u. path. Anat., 35:314, 1924-25; 

 Schultz, A., and Lohr, G.: Zentralbl. f. allg. Path. u. path. Anat., 36:529, 

 1925. 



