100 Microscopic Histochemistry 



the Liebermann-Burchardt test, which is positive with all 

 unsaturated sterols,^^ whether esterified or not. For all prac- 

 tical purposes, it may be considered a specific test for choles- 

 terol and its esters. 



Method 



Mordant frozen sections in 2 per cent ferric alum for 24 

 hours. This step is essential, although its chemical back- 

 ground is not well understood. Rinse sections in distilled 

 water. Mount them on slides, blot them dry. Place a few 

 drops of a mixture of equal parts of glacial acetic acid and 

 concentrated sulfuric acid (caution: cool test tube while 

 mixing the acids!) on the section and cover it with a cover 

 shp. A change of colors from purple-red through dark blue 

 to blue-green will take place within about 1 minute. Only 

 the last shade mentioned is diagnostic for cholesterol. 



h) The digitonin reaction^*^ is specific for unesterified 

 3-cis-OH sterols, such as cholesterol, the vitamin D com- 

 pounds, isoandrosterone, etc.; 3-trans-OH compounds (an- 

 drosterone, bile acids) do not react. 



Method 



Immerse frozen sections for a few hours in a 0.5 per cent 

 solution of digitonin in 50 per cent alcohol. Wash in 50 per 

 cent alcohol and in water. Mount in glycerin- jelly. Under the 

 polarizing microscope typical groups of fine needle-shaped 

 crystals are seen; they are birefringent. 



C ) For car otenoids.— One of the carotenoids, vitamin A, 

 has been mentioned under "Fluorescence microscopy." Ca- 

 rotenoid pigments will be considered in the chapter on 

 "Pigments." 



35. Sobotka, H.: The chemistry of the sterids (Baltimore: Williams & 

 WilHns, 1938), p. 158. 



36. Brunswik, H.: Ztschr. f. wissensch. Mikr., 39:316, 1922; Leulier, A., 

 and Noel, R.: Bull, d'histol. appliq. a la physiol., 3:316, 1926; Leulier, A., 

 and Revol, L.: Bull, d'histol. appliq. a la physiol., 7:241, 1930; Lison, L.: 

 Histochimie animale (Paris: Gauthier-Villars, 1936). 



