STAINING, PRACTICAL AND THEORETICAL 



Technique: 



1. Immerse pieces of fresh tissue in solution A for two days or 

 longer: longer fixation, up to a month is apparently not harmful. 



2. Wash thoroughly in running tap water. 



3. Decalcify (if necessary) in solution B. 



4. Wash thoroughly in running tap water. 



5. Infiltrate with 2% gelatine for two to three days at 37° C. 



6. Transfer to 5% gelatine and leave therein at 37° C. for the 

 same length of time. 



7. Immerse in 10% gelatine at 37° C. for the same length of time. 



8. Transfer the dish of 10% gelatine containing the block to 

 the refrigerator and leave it to chill for several hours. 



9. Trim the block, by cutting away excess gelatine, then place 

 it in cold McManus fluid (solution A) for twenty-four hours to 

 harden the gelatine. 



Note: Embedded specimens can be stored in this fluid at room 

 temperature. 



ID. Cut thick serial sections, at an average thickness of 75ju., 

 on a freezing microtome, and orient them on slides of suitable 

 dimensions, on a warm stage, in a few drops of 1% gelatine. 



1 1 . Allow the excess fluid to evaporate, then place the slides in 

 ice-cold McManus fluid in a grooved staining dish and transfer 

 the whole to a refrigerator to harden the gelatine. 



12. Wash the slides in cool, running tap water, then in distilled 

 water, then transfer to 70% alcohol. 



13. Immerse in freshly filtered Sudan black solution for fifteen 

 to thirty minutes. 



14. Drain the slides well. 



15. Differentiate in two successive baths of 50% alcohol: this 

 step takes anything from a few seconds to a few minutes and is 

 best controlled by microscopic examination. 



16. Wash well in water, then mount in glycerine jelly: allow 

 the edges of the coverslips to dry thoroughly, seal with a non- 

 aqueous cement such as Laktoseal. Alternatively, Aquamount 

 may be used instead of glycerine jelly, in which case ringing of 

 the coverslips is not necessary. 



Results: 



Myelin stains bright green to greyish green in thick sections as 

 above. Large nerves and the smaller myelinated axons are well 



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