Acid Phosphatase M?) 



solution: 



Incubating solution: 



sodium a-naphthyl phosphate 10-20 mg. 



O.l M veronal acetate buffer, pH 9.2 20 ml. 



stable diazotate of 4-benzoyl amino-2:5-dimeth- 



oxyaniline (fast blue RR) 20.0 gm. 



procedure: 



1. Filter enouoh incubating; sokition onto slides to cover section.s 

 adequately. Incubate, room temperature: 15-60 minutes. 



2. Wash in running water: 1-3 minutes. 



3. Counterstain, Mayer's hematoxylin: 4-6 minutes. 



4. Wash in rimning water: 15-30 minutes. 



5. Mount in glycerol jelly. 



results: 

 sites — black 



comments: 



Alcohol fixation can be used, but is of no great advantage for frozen 

 sections. 



References: Ciomori (1951), Manheimer and Seligman ( 1949), and 

 Pearse (1960) 



Acid Phosphatase 



Most methods lack uniformity and some loss occurs during fixation 

 and preparation for sectioning. Gomori (1936) considered the lead sul- 

 fide method "capricious," prodticing artifacts, and that the benzoyl- 

 naphthol phosphate post-coupling method of Rutenburg and Seligman 

 (1935) was best. Since then Burstone's (19393) method has appeared. 

 Both of the latter are included. 



fixation: 



Rutenburg and Seligman Method: 



1. Cold neutral 10% formalin: 24-48 hours (neutralize with NaCl). 

 Wash in 0.857^ NaCl 2 changes: 30 minutes. 



Cut frozen sections. 



2. Fresh frozen sections (6-10 microns) cut in cryostat, mounted on 

 uncoated slides, air-dried. 



