64 Microscopic Histochemistry 



acting substances (glycogen and mucin) and carbohydrates 

 of the connective tissue. 



Staining with Schiffs reagent —StSiin sections for about 

 10-15 minutes in SchiE's reagent (straight or diluted with 

 an equal volume of water ) . Usually very little staining is seen 

 during this step, especially after chromic acid oxidation. 

 Rinse the reagent oflF; flood sections for 1 or 2 minutes with a 

 1-3 per cent solution of Na bisulfite. Wash under the tap for 

 about 5-10 minutes. It is during this washing that the stain- 

 ing of mucin, glycogen, etc., in an intense purplish-red shade 

 becomes more apparent. Counterstain with hematoxylin 

 (staining after chromic acid often rather poor). Dehydrate 

 and mount. 



Staining with meihenamine-siher.—Rmse sHdes thoroughly 

 in distilled water. Incubate them at a temperature of 37°- 

 50 °C. in a mixture consisting of 25 ml. each of methenamine 

 stock solution and distilled water with a few ml. of an M/5 

 borate buffer of pH 8.3-9.2 added. The more alkaline the so- 

 lution, the faster it will work but the more likely it is to pro- 

 duce a brownish background, especially in the case of unduly 

 long incubation. The reaction will show up in a yellowish- 

 brown shade in about 1 hour. Inspect sections under the mi- 

 croscope once every 30 or 60 minutes; as soon as glycogen, 

 mucin, etc., appear in black or in a very dark purpHsh-brown 

 shade while the background is still a very light tobacco 

 brown, remove sections from the silver solution, rinse in dis- 

 tilled water, and tone with a 0.1-0.2 per cent solution of gold 

 chloride for about 10 minutes. This treatment usually com- 

 pletely bleaches the background. Remove unreacted silver 

 by a short rinse with dilute (1-3 per cent) Na thiosulfate. 

 Wash and counterstain as desired (e.g., with hematoxylin 

 and eosin). Dehydrate and mount. 



If the background is too dark, carefully differentiate the 

 section (before gold toning) in a mixture containing about 

 0.2 per cent of ferric sulfate and 0.5 per cent of sulfuric acid 



