ACID POLYSACCHARIDES 45 



If the periodate and iodate are not washed out they will give rise to a 

 brownish coloration in the Feulgen reagent. 



Control sections or smears may be made by placing in 70% alcohol (or 

 water), instead of in the periodic acid soln. A or B, and then carrying 

 through the remaining steps without change. 



Egg albumin adhesive may take a slight stain due to the carbohydrate 

 content of this material. 



If neutral polysaccharides are to be stained, a counterstain with a basic 

 dye (such as 0.02 mg. malachite green per ml. water) may be used. If 

 mucin or acid polysaccharides are to be stained, the counterstain should be 

 an acid dye. 



ACID POLYSACCHARIDES — HYALURONIC ACID* 



For the demonstration of acid polysaccharides of the hyaluronic 

 acid type Hale (1946) employed fixation in a dehydrating medium 

 to prevent solution of the water-soluble acid polysaccharide, com- 

 bination of the latter with iron, and demonstration of the iron by 

 means of the Prussian blue reaction. The iron will not combine with 

 neutral polysaccharides or proteins according to Hale. In order to 

 differentiate between hyaluronic acid and other substances which 

 might give the blue stain, Hale suggests that hyaluronidase be 

 used to digest away the hyaluronic acid in the section on one slide 

 and a comparison be made to an undigested section. 



Hale Method for Acid Polysaccharides 



SPECIAL REAGENTS 



Acetic-Iron Solution. Mix equal vol. dialyzed ferric hydroxide, 

 concentration not stated (Hale used the product of the British 

 Drug Houses Ltd.), and 2 M acetic acid. 



0.02 M Potassium Ferrocyanide in 0.14 M Hydrochloric Acid. 



PROCEDURE 



1. Fix 3-4 mm. pieces of tissue in Carnoy fluid (6 vol. absolute 

 alcohol, 3 vol. chloroform, and 1 vol. glacial acetic acid) for 0.5 hr. 



2. Treat with absolute alcohol; clear, and mount in paraffin. 



3. Prepare sections and place on slides without albumin adhesive. 



4. Flood the deparaffinized sections with the acetic-iron soln., 

 and after 10 min. wash well with distilled water. 



5. Treat the sections with the ferrocyanide soln. for 10 min. and 



* See page 46 for the staining of mucoproteins. 



