208 Silver ImpregnaLion II (chap. 15) 



Decfeneratinff Axons 



Nauta and Gygax (1954) S 



fixation: perfusion method seems to be preferred. 

 Anderson (19'>9): 



1. Perfuse with 0.9% sodium chloride (intracardiac and intra-aortic 

 canulation) until fluid escaping is clear. 



2. Perfuse with 500 ml. of 5% potassium dichromate and 2.5% po- 

 tassium chlorate. This is peculiar to Anderson; others perfuse 

 with the fixino solution. 



3. Remove tissue and fix in 10% formalin neiuralized with carbon- 

 ate: 1 week or longer. Nauta and Gygax recommend 1-3 months 

 as best. 



EMBEDDING AND SECTIONING: 



1. If embedding is necessary use gelatine. Wash slices (5-10 mm. 

 thick) in running water: 24 hours. Incubate in 25% gelatine, 37 °C 

 (cover tightly): 12-18 hours. Wipe off excess gelatine and immerse 

 in cool formalin: 6 hours or longer. 



2. Frozen sections. Cut sections 15-25 microns and place in 10% 

 formalin, room temperature or cooler. Can be stored in formalin, 

 preferably in refrigerator. Process only a few sections (6-10) at 

 one time. 



Adey et a I. (1958) prefer a dual-freezing block method, using dry 

 ice evaporated in 70% alcohol, over CO2 freezing. This produces 

 more even freezing. Rapid freezing and tha^ving with CO2 may rup- 

 ture some of the fibers (Freon equipment eliminates some of this 

 problem). 



solutions: 



Ammoniacal silver nitrate: 



silver nitrate 0.9 gm. 



distilled water 20.0 ml. 



When dissolved add: 



absolute ethyl alcohol 10.0 ml. 



ammonia, concentrated 1.8 ml. 



sodium hydroxide, 2.5% aqueous 1.5 ml. 



Reducing fluid: 



distilled water 400.0 ml. 



absolute ethyl alcohol 45.0 ml. 



