92 MICROSCOPIC TFX'HNIQUES 



activity in histological preparations. Sharlit et al. (1942) have 

 pointed out that the method for demonstrating dopa oxidase may 

 of itself, in the absence of substrate, cause an increase in melanin. 

 This makes it necessary to run suitable control experiments. The 

 reaction may be hastened by employing a buffer of a little higher 

 pH, or retarded by shifting toward the acid side. 



Laidlaw Melliod for Dopa Oxidase 



SPECIAL REAGENTS 



Dopa Stock Solution. Dissolve 0.3 g. dopa (Hoffmann-La Roche, 

 labeled "for Bloch's dopa reaction") in 300 ml. cold distilled 

 water. Store in a refrigerator and discard when a distinct red color 

 has developed. 



Buffered Dopa Solution (pH 7.4) . Add 2 ml. potassium dihydrogen 

 phosphate (9 g. KH2PO4/I.) and 6 ml. disodium hydrogen phos- 

 phate (11 g. Na2HP04.2H20/l.) to 25 ml. dopa stock soln. Filter 

 through fine paper. 



Buffer Solution for Control Experiment. Replace the 25 ml. dopa 

 soln. with an equal vol. distilled water in the buffered dopa soln. 

 above. 



PROCEDURE 



1. Prepare frozen sections of fresh tissue. (It is stated that tissue 

 hardened in 5% formalin for 2-3 hr. may be used.) 



2. Rinse in distilled water for a few sec. and transfer at once to 

 the buffered dopa soln. At 30-37° the soln. becomes red in about 

 2 hr. and sepia brown in 3-4 hr. Do not let the sections remain in 

 the soln. once it becomes sepia colored since overstaining may result. 

 Examine from time to time under a microscope to determine the 

 proper intensity of staining. It is good practice to change to a fresh 

 dopa soln. after the first 30 min. 



3. Wash sections in distilled water, dehydrate, and counterstain 

 w^ith alcoholic cresyl violet or methyl green-pyronine. 



4. Clear and mount in balsam. 



5. Run controls by treating tissue as above with the substitution 

 of buffer soln. for the buffered dopa soln. in step 2. 



