HISTOCHEMISTRY OF PLACENTA 



889 



sis of the phenomenon of metachromasia 

 was made recently by Bergeron and Singer 

 (1958). 



B. CARBOHYDRATES 



Alodcni liit^toc'hemistry of carbohydrates 

 revolves principally around the periodic 

 acid-Schiff (PAS) reaction developed by 

 McManus (1945) and Hotchkiss (1948). 

 This procedure involves the oxidation by pe- 

 riodic acid of 1,2-glycol linkages which are 

 common in sugars. The resulting aldehydic 

 groups are colored by the Schiff's reagent. 

 The i)eriodic acid-Schiff reaction also stains 

 other carbohydrate containing substances 

 including glycoproteins, mucoproteins, and 

 glycolipoproteins (Leblond, 1950). Glyco- 

 gen can be differentiated histochemically 

 from these substances by the use of saliva 

 or malt diastase on control sections. In ear- 

 lier studies of the placenta, glycogen storage 

 was assessed by Best's carmine stain as well 

 as l)y means of alkaline silver nitrate (Mit- 

 chell and Wislocki, 1944) . Also in much of 

 the earlier work a digested control section 

 was not used. 



Sudan dyes, which dissolve readily in tri- 

 glycerides, are most commonly used to dem- 

 onstrate these lipids. Sudan black B has su- 

 l)erseded Sudan III and IV by virtue of its 

 moi'e favorable color and also because it re- 

 veals many lipids in addition to triglycer- 

 ides. Mitochondria are stained by Sudan 

 black B presumably through their phospho- 

 lipid content. There is some evidence that 

 staining by Sudan black occurs also through 

 chemical binding (Pearse, 1960). Osmium 

 tetroxide is also employed for demonstrat- 

 ing lipid, although it is less specific than 

 Sudan staining. This energetic agent, which 

 is the principal fixative-stain for electron 

 microscopy, will oxidize unsaturated com- 

 pounds turning them grey or black. For the 

 demonstration of phospholidips, Baker 

 I 1 946 ) introduced an acid hematein test. 



A group of histochemical reactions is 

 characteristic of the lipid droplets of the 

 xferoid-producing organs. Besides staining 

 intensely with Sudan black B, these lipid 

 droplets, in frozen sections of formalin-fixed 

 material, react positively in tests for car- 

 bonyl groups, such as the Schiff test and 



hydrazine methods ( phenylhydrazine 

 method of Bennett (1940) and the naph- 

 thoic acid hydrazide method of Ashbel and 

 Seligman (1949)). They are fluorescent 

 when viewed with ultraviolet light and con- 

 tain birefrigent crystals (Dempsey and 

 AVislocki, 1944; Wislocki and Dempsey, 

 1946a, b; Kockenschaub, 1952). The drop- 

 lets also give colored products when treated 

 with mineral acids, as in the Liebermann- 

 Burchardt reaction or the Schultz reaction 

 for cholesterol. All of the above reactions 

 are prevented following extraction of the 

 sections with acetone at room temperature. 



In the ])ast it was proposed that all or 

 some of these reactions might be given by 

 ketosteroids, i.e., by hormones and their 

 immediate precursors (Dempsey and Wis- 

 locki 1946). Seligman and Ashbel (1951) 

 adduced evidence for the ketonic nature of 

 some of the carbonyls and for the specificity 

 of the naphthoic acid-hydrazide reaction in 

 the steroid hormone-producing glands. How- 

 ever, it was gradually realized that none of 

 the above reactions is specific for ketoster- 

 oids, and a reevaluation of their significance 

 was undertaken by Deane and Seligman 

 (1953). More recently Karnovsky and 

 Deane (1954, 1955) have shown by both 

 chemical and histochemical means that the 

 carbonyl grouj^s in adrenal cortical lipids 

 seem to be exclusively aldehydes produced 

 by the auto-oxidation of unsaturated fatty 

 acids during fixation. The reaction is inhib- 

 ited by the addition of various anti-oxidants 

 and chelating agents to the fixative. More- 

 over, hydrazine reactivity occurs only when 

 there is reactivity to the Schiff reagent, and 

 the appearance of aldehydes is correlated 

 with the disap])earance of double bonds in 

 the lipids. 



On the other hand, the experimental ob- 

 servations of Kai'iiovsky and Deane proved 

 rather conclusively that the percentage of 

 unsaturated fatty acids which are the source 

 of the aldehyde groups is high in function- 

 ally active adrenals and declines in inac- 

 tive glands, thus confirming the earlier 

 assertions (Bennett, 1940; Wislocki and 

 Bennett, 1943; Dempsey and Wislocki, 

 1946) that the intensity of the carbonyl re- 

 action correlates well with secretory activ- 

 ity. 



