1 



Negri Bodies {Rabies) 829 



entire process can be handled in 8 hours, inchiding fixing embedding, 

 and staining. 



Massignani and Malferrari (1961) Method 



fixation: 10% formalin or saturated aqueous mercuric chloride-abso- 



hue alcohol (1:2). 

 EMBEDDING and SECTIONING: paraffin method, 4 microns. 



solutions: 



Harris hematoxylin, see page 125. 

 Dilute hydrochloric acid: 



hydrochloric acid, concentrated 1.0 ml. 



distilled water 200.0 ml. 



Dilute lithium carbonate: 



lithium carbonate, saturated aqueous 1.0 ml. 



distilled water 200.0 ml. 



Phosphotimgstic acid-eosin stain: 



Grind together 1 gm. eosin Y (C.I. 45380) and 0.7 gm. phospho- 

 ttnigstic acid. Mix thoroughly into 10.0 ml. of distilled water and 

 then bring vokuiie up to 200.0 ml. with distilled water. Centrifuge at 

 1,500 rpm: 40 minutes. Pour off supernatant solution but do not 

 throw it away. Dissolve the precipitate in 50 ml. of absolute alcohol. 

 When dissolved, add to the stipernatant solution. The soltition is 

 ready to use. If the eosin and phosphottmgstic acid are not ground 

 together and then dissolved it reqtiires 24 hours for the dye-mordant 

 combination to form. 



PROCEDURE: 



1. Deparaffinize and hydrate sections to water, remove HgCU. 



2. Stain in hematoxylin: 2 minutes. 



3. Wash in running water: 5 miniues. 



4. Dip 8 times in dilute hydrochloric acid. 



5. Wash in running water: 5 minutes. 



6. Blue in dilute lithium carbonate: 1 minute. 



7. Wash in running water: 5 mintites. 



8. Dehydrate to absoliue alcohol. 



9. Stain in phosphotimgstic acid-eosin: 8 minutes. 



10. Rinse in distilled water. 



11. Dehydrate by quick dips in bO%, 70%, 80%,, and 907^ alcohol, 

 then follow with 95% alcohol: 1 second. 



12. Complete dehydration in absolute alcohol: 2-3 changes: 4 min- 

 utes each. 



13. Clear and moiuit. 



