80 MICROSCOPIC TECHNIQUES 



lute acetone and fix for 12-24 hr. in a refrigerator. Dehydrate at 

 room temperature in two changes of absolute acetone for 6-12 hr. 

 each time. 



2. Optional step. To strengthen sections which tend to break up 

 when floated on the lukewarm water after sectioning, impregnate the 

 tissue with the acetone-acetylcellulose soln. 24 hr. 



3. Drain off the fluid rapidly and place in two changes of benzol 

 for 30 min. each. 



4. Embed in paraffin not over 56° up to 2 hr. To hasten the proc- 

 ess use 3 changes of paraffin, each for 20 min., and carry out the 

 second change in vacuo in a wide-mouth bottle with a one-hole 

 rubber stopper fitted with a glass tube. Connect the glass tube by 

 rubber tubing, passed through an air hole in the paraffin oven, to a 

 water aspirator via a safety bottle. 



5. Cut sections 4-8 ju, thick, fioat them on lukewarm water 

 (30-35°), and mount on slides. 



6. Let slides dry, place in the paraffin oven for 10 min. to melt the 

 paraffin, and run through xylol and alcohols to distilled water. Re- 

 move preformed mineral deposits as described on page 78. 



7. Incubate the sections for 1-2 hr. at 37° in the substrate me- 

 dium. 



8. Rinse with water, immerse in the cobalt soln. for 5 min., and 

 rinse well with several changes of distilled water. 



9. Place in the diluted ammonium sulfide soln. for 1-2 min. 



10. Wash well in water, counterstain if desired, dehydrate, and 

 mount. 



Result. Sites of the phosphatase activity appear brown or black. 



ACID PHOSPHATASE* 



The staining method for alkaline phosphatase cannot be used for 

 acid phosphatase since calcium phosphate is soluble at a pH around 

 5, which is optimum for the action of the latter enzyme. Hence, 

 Gomori (1941b) employed lead ions in the substrate medium at pH 

 4.7 so that insoluble lead phosphate would be formed at the sites of 

 enzymatic activity. The lead phosphate was then converted either 

 to brown or black lead sulfide, or was stained a purplish-red with 

 acridine red. Wolf, Kabat, and Newman (1943) introduced several 



*See Bibliography Appendix, Refs. 1 and 9. 



