Myelin 215 



3. Wash in distilled water, 3 changes: 10 minutes. 



4. Impregnate, 37°C: overnight. 



5. Shake off superfluous Huid and place in reducer: 2 minutes. 



6. Wash in rimning water: 3 minutes; rinse in distilled water. 



7. Tone in gold chloride, 0.2% (0.2 gm./lOO ml. water): 3 minutes. 



8. Rinse in distilled wMter. 



9. Treat with oxalic acid, 2% (2 gm./lOO ml.): 3-10 minutes; when 

 axons are thoroughly black, remove. 



10. Rinse in distilled water. 



1 1. Fix in sodium thiosulfate, 5% (5 gm./lOO ml.): 3 minutes. 



12. Wash in rimning water: 10 minutes. 



13. Rinse briefly in 95% alcohol. 



14. Stain in Luxol fast blue: overnight. 



15. Rinse in 95% alcohol; rinse in distilled water. 



16. Treat wkh lithium carbonate, 0.05% (.5 gm./lOOO ml. water): 

 15 seconds. 



17. Differentiate in 70% alcohol: 20-30 seconds. 



18. Rinse in distilled water. 



19. Repeat steps 16 and 17 if necessary. (The author finds that usu- 

 ally 3 repeats is necessary for sharp differentiation.) 



20. Dehydrate, clear, and moimt. 



results: 



axis cylinders — black 

 myelin sheaths — green blue 



comments: 



Margolis and Pickett write that slides may be left in step 6 until time 

 to prepare them for Luxol fast blue staining. This tends to encourage 

 loose sections. The author has lost no sections by carrying the slides 

 through to step 13. Transfer them into absolute alcohol and coat 

 them with nitrocellulose. Harden the nitrocellulose and store the 

 slides in 70-80% alcohol until ready to stain in luxol fast blue. Slides 

 have been left in this condition for over the weekend with completely 

 satisfactory results. The nitrocellulose protection also permits freer 

 agitation of slides in the differentiating fluids than without this coat- 

 ing. 



Ora's Method (1958) S 

 fixation: 10% formalin. 



section: at 17 microns, paraffin method. 



