SECTION TWO 



Technique: 



Material should be fixed in io% formalin. 



Paraffin or frozen sections give somewhat better results than 

 Celloidin. Affixed Celloidin give better results than loose Cel- 

 loidin sections. 



(a) Frozen sections 



1. Cut sections 25^11 in thickness and place them in distilled 

 water. 



2. Immerse in 70% alcohol for ten to fifteen minutes. 



3. Stain from five to twenty-four, but preferably not less than 

 sixteen hours, in the Luxol fast blue solution, in a stoppered jar 

 in an oven at 40° C. 



Note: For staining four sections of the brain stem of a monkey, for 

 example, 20 to 25 ml. of the stain should be used and then discarded. 



4. Immerse in 95% alcohol and wash off the excess stain. 



5. Wash in distilled water. 



6. Immerse for two or three seconds, but no longer, in the 

 lithium carbonate solution, as the first stage of differentiation. 



7. Continue the differentiation in several changes of 70% 

 alcohol until the grey and white matter can be distinguished, but 

 taking care not to over- differentiate. 



8. Wash in distilled water. 



9. Immerse in the lithium carbonate solution for three to five 

 seconds, but no longer. 



10. Complete the differentiation by immersing in several 

 changes of 70% alcohol, until the white matter is stained greenish- 

 blue in sharp contrast with the colourless grey matter. 



11. Wash thoroughly in distilled water. 



12. Stain for one to two minutes in the cresyl fast violet solution, 

 which should be warmed carefully and filtered before use. 



13. Wash for two or three seconds in distilled water. 



14. Differentiate in several changes of 95% alcohol until colour 

 ceases to come away from the preparation and the alcohol is no 

 longer tinted. 



15. Clear in xylol-terpineol (Solution D). 



16. Clear in xylol and mount. 



H5 



