SECTION TWO 



For nervous tissues (Benda's method) 



Solutions required: 



A. Nitric acid, cone. . . . . i volume 

 Distilled water lo volumes 



B. Potass, dichromate 2% 



C. Chromic acid i% 



D. Iron alum 4% 



E. Alizarin Red, S, saturated in abso- 



lute alcohol . . . . . . I ml. 



Distilled water . . . . . . 90 ml. 



F. Toluidine Blue o-i% aqueous 



Technique: 



1. Material is fixed in 90-95% alcohol for at least two days. 



2. Pieces, which must not be thicker than 0-5 cm., are immersed 

 in Solution A for twenty-four hours. 



3. Transfer to Solution B for twenty-four hours. 



4. Transfer to Solution C for forty-eight hours ; then wash in 

 water for twenty-four hours. 



5. Dehydrate in the usual manner. 



6. Clear in beechwood creosote for twenty-four hours ; then in 

 benzol for twenty-four hours. 



7. Embed in paraffin wax via four graded mixtures of paraffin 

 wax and benzol, the first at room temperature, the second at 

 38° C, the third at 42° C. and the fourth at 45° C. 



8. Mount sections on sHdes: bring down to distilled water, 

 mordant sections on slides with Solution D for twenty-four hours, 

 then wash thoroughly in water. 



9. Stain for two hours with Solution E; then rinse in tap water. 



10. Flood slides with Solution F and warm gently until vapour 

 is given ofi^; or stain at room temperature for 24 hours. 



11. Rinse in 1% acetic acid; then dry by blotting carefully. 



12. Pass through absolute alcohol; then differentiate for about 

 ten minutes in beechwood creosote; dry by blotting carefully, 

 wash with xylol, and mount. 



Reference: Benda, C. (1900, 1901). 



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