STAINING, PRACTICAL AND THEORETICAL 



NAPHTHANIL DIAZO BLUE-H ACID 

 For histidine 



Solutions required: 



A. Gram's iodine . . . . • • 30 ml. 

 Ammonia solution (sp. gr. o-88o) 2 ml. 



B. Veronal acetate buffer pH 9-2 . . 200 ml. (approx.) 



C. Solution B . . . . • • 50 ml. 

 Naphthanil diazo blue B • • 0'05 gm. 



(Michrome blue salt 250) 



D. H acid (8-amino-i-naphthol-3:6- 



disulphonic acid) . . . . i gm. 



Solution B . . . . . . 50 ml. 



Technique: 



1. Fix paraffin sections of 10% formalin-fixed tissues to slides. 



2. Dewax with xylol and pass through the usual graded alcohols 

 into distilled water. 



3. Immerse in solution A at room temperature for twenty- four 

 hours. 



5. Rinse in water. 



6. Rinse in 96% alcohol until the sections lose their yellowish 

 colour. 



2. Rinse in water, then in solution B (veronal- acetate buffer). 



8. Immerse in reagent C for fifteen minutes at 0° C. to 4° C. 



9. Wash in water. 



10. Immerse for two minutes in each of three changes of the 

 veronal-acetate buffer (solution B). 



11. Immerse in solution D at 0° C. to 4° C. for fifteen minutes, 

 agitating the slides gently at intervals. 



12. Wash in running water. 



13. Dehydrate with two or three changes of anhydrous acetone 

 or rapidly in absolute alcohol. 



14. Clear in xylol and mount in Cristalite, Clearmount, D.P.X. 

 or Emexel. 



Results: 



Sites of histidine groups are brick-red to reddish-brown. 



References : 

 Landing, B. H. & Hall, H. E. (1956). 

 Gurr, E. (iQSM- PP- i5-i9- 



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