196 Microscopic Histochemistry 



cent solution of lead nitrate to 50 ml. of a 0.05 M maleate 

 buffer of pH 5.5-5.6; shake until the initial white precipitate 

 dissolves. Add a few drops of a 0.2 M solution of MnCl2 and 

 1.5-2 ml. of the phosphonate stock solution. If the mixture 

 becomes turbid, place it in the paraffin oven and keep it 

 there for 10-30 minutes or until the turbidity settles. Filter it 

 into a Coplin jar. Incubate sections for 12-24 hours, at 37° C. 

 Since the substrate is not entirely stable at pH 5.5 but de- 

 composes slowly into free phosphate and p-chloroaniline, the 

 Coplin jar must be supported in an inclined position, with 

 the tissues facing downward, to avoid, as far as possible, the 

 indiscriminate precipitation of lead phosphate all over the 

 tissues. This maneuver will cause the heavy precipitate to 

 settle on the back surface of the slides. 



Remove slides from the incubating mixture, wipe precipi- 

 tate from back surface. Rinse them thoroughly in distilled 

 water. Remove superficial precipitate by moving the slides 

 around in a 0.1 M citrate buffer of pH 4.5-5. As soon as the 

 glass appears completely clear around the tissue, rinse slides 

 under the tap. This is the most delicate step in the entire 

 procedure. Insufficient differentiation will leave a coarse 

 black precipitate in the finished section, while overdifferen- 

 tiation may remove part or all of the lead phosphate depos- 

 ited by enzymatic action. 



Treat slides with ammonium sulfide, etc., as in the method 

 for acid phosphatase. 



In its present form the method is not entirely dependable. 

 The difficulties are essentially the same as with the acid 

 phosphatase technique (lack of uniformity in staining, some- 

 times even in the case of consecutive serial sections; unex- 

 plained failures to get a positive reaction). 



Moderate amounts of the enzyme are found in practically 

 all animal tissues; very high concentrations can be demon- 

 strated in the vast majority of maUgnant epithehal neo- 

 plasms. As a rule, the intensity of the reaction roughly par- 

 allels the degree of histological malignancy. Polyps of the 



