Enzymes 191 



especially a high temperature of the paraflBn oven and long 

 exposure of the tissue to hot paraflBn seem to be harmful. 



According to several authors (Hard and Lassek,^^ Doyle J^ 

 Goetsch and Reynolds"^^), the enzyme is gradually inacti- 

 vated on storage of paraflBn blocks, ribbons, or mounted 

 sections. 



Doyle asserts that lead ions inhibit the enzyme very mark- 

 edly (about 85 per cent). Using the regular histochemical 

 substrate mixture, the writer found about 35 per cent inhibi- 

 tion by lead in test-tube experiments. 



2. The use of unfixed or partially fixed, unembedded tis- 

 sue. Frozen sections may show a relatively high activity, but 

 the aflBnity of undenatured proteins to lead salts is likely to 

 produce false positive reactions. Lead adsorbed by paraflBn- 

 embedded tissues can be washed out readily by dilute acetic 

 acid (exception under 4); however, frozen sections of un- 

 fixed or acetone-fixed tissues will hold lead so stubbornly that 

 even prolonged washing in strong acetic acid cannot remove 

 it completely. Formalin-fixed protein has much less aflBnity 

 to lead. 



3. Failure to use an acid rinse after incubation. Even de- 

 natured proteins may adsorb some lead from the substrate 

 mixture but will release it easily when rinsed in dilute acetic 

 acid. The acid rinse is an important step; it removes protein- 

 bound lead but leaves enzymatically produced lead phos- 

 phate untouched. If it is omitted (as it apparently was by 

 Newman, Kabat, and Wolf^^ and by Takeuchi and Ta> 

 noue^^), the result will be a nonenzymatic staining of the 

 background, mainly of nuclei. . 



4. Certain structures such as axons retain a high aflBnity 

 to lead even after paraflfin-embedding; in fact, axons can be 



70. Doyle, W. L.: Proc. Soc. Exper. Biol. & Med., 69:43, 1948. 



71. Goetsch, J. B., and Reynolds, P. M.: Stain Technol., 26:145, 1951. 



72. Newman, W., Kabat, E. A., and Wolf, A.: Am. J. Path., 26:489, 

 1950. 



73. Takeuchi, T., and Tanoue, M.: Kumamoto M. J., 4:41, 1951. 



