90 MICROSCOPIC TECHNIQUES 



9. Rinse in repeated changes of distilled water and immerse in 

 the diluted ammonium sulfide soln. for 1-2 min. 



10. Wash well in water, counterstain lightly with hematoxylin 

 and eosin, dehydrate in alcohols, clear in gasoline or tetrachloro- 

 ethylene, and mount in Clarite dissolved in the same liquid. (Toluol 

 or xylol causes fading of the stain.) 



Result. Sites of lipase activity appear golden brown. 



PEROXIDASE 



Most of the various microchemical methods for the histological 

 localization of peroxidase activity are based on the oxidation of 

 benzidine. The methods of McJunkin ( 1922) , designed for use with 

 human tissues, and Armitage ( 1939), developed for examining blood 

 and bone marrow smears, have been chosen for presentation here 

 since they are the most recent and seem to be the best. Peroxidase 

 actually occurs most abundantly in plants, but the methods appear 

 to have been worked out for animal tissues or cells exclusively. 

 However, there appears to be no reason why these methods cannot 

 be adapted to plant material as well. 



McJunkin Method for Peroxidase in Tissue Sections 



SPECIAL REAGENTS 



Benzidine Reagent. Dissolve 100 mg. benzidine in 25 ml. 80% 

 methanol and add 2 drops 3% hydrogen peroxide. Dilute with 

 1-2 vol. distilled water before using. Store in the dark. 



PROCEDURE 



1. Place formalin-fixed tissue, in pieces 1 mm. thick, in 70% 

 acetone for 1 hr. followed by pure acetone for 30 min., benzol for 

 20 min., and melted paraffin 20 min. 



2. Cut sections 3.5 to 5.0 [i, fix to slides with albumin, and dry 

 overnight at room temperature. 



3. Remove paraffin by placing in benzol for 20 sec. and acetone 

 for 10 sec. 



4. Plunge in water for a few sec. and remove excess water. Apply 

 the benzidine reagent for 5 min. and transfer to water for 5 min. 



5. The sections may be stained with Harris hematoxylin for 2 

 min., rinsed in water 1 min., and stained with 0.1% eosin for 20 sec. 



