202 Silver hnpregnation II (chap. 15) 



3. Wash in distilled water: 2-6 hours depending on size of bhxk. 

 Change every half hour. 



4. Impregnate with 1-1.5% aqueous silver nitrate (1.0-1.5 gm./lOO 

 ml. water): 3 days, 37 °C. 



5. Wash in distilled water: 20 minutes to 1 hour, depending on size. 

 Change every 10 minutes or use a large volume of water and 

 shake every 10 minutes. Periphery and central portions must be 

 equally stained. 



6. Impregnate in ammoniated siher solution: 6-24 hours. 



7. Wash in distilled water: 15 minutes to 1 or 2 hours, depending 

 on size. 



8. Reduce in 1% formalin: 6-12 hours. 



9. Wash in running water: 10-15 minutes. 



10. Dehydrate, clear and embed. Section and mount. 



11. Deparaffinize, clear, and cover. 



Alternate method, for oold toning: 

 (fl) Hydrate sections to water. 

 {b) Gold tone and fix. 

 (c) Dehydrate, clear, and moimt. 



results: 

 /nerve fibers, neurofibrils — brown to black (no gold toning) 



— grey to black (with gold toning) 



Fluorescent Method (zeiger et al., 1951) S 

 fixation: 95% ethyl alcohol. 



sectioning: paraffin method. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Stain in 0.1% acridine orange (C.I. 46005): 6 minutes. 



3. Differentiate in 95% ethyl alcohol: 2 seconds. 



4. Blot with filter paper and mount in fluorescent mountant. 



results: 



unmyelinated fibers — bluish gray 

 myelinated fibers — brownish orange 



comments: 



Fresh tissue can be cut frozen and stained in acridine orange made 

 up in physiological saline (page 410) or in Ringer's solution (page 

 411). 



