SECTION TWO 



Technique: 



1. Fix material in Lavdowsky's mixture or in io% formalin 

 and embed in paraffin wax. 



2. Stain in Harris, Ehrlich or Delafield Haematoxylin for five 

 to ten minutes. 



3. Blue in tap water or 1% lithium carbonate solution. 



4. Stain in the azophloxine solution for two minutes. 



5. Rinse in 0-2% acetic acid. 



6. Stain in the orange G-fast green solution for one minute. 



7. Differentiate for about five minutes with 0-2% acetic acid. 



8. Rinse in distilled water. 



9. Remove excess distilled water by draining and blotting round 

 the edges of the sections carefully, but do not allow to dry com- 

 pletely. 



10. Dehydrate directly with two or three changes of absolute 

 alcohol, or with cellosolve. 



Note: Absolute and 70% alcohol should be avoided as they have 

 a tendency to remove the azophloxine. 



Results: 



Connective tissue, green. Striated muscle, brick red. Smooth 

 muscle, reddish violet. Nerves, blue-grey. Ganglion cells, violet. 

 Erythrocytes, orange. Cardiac conductive tissue is easily dis- 

 tinguishable from cardiac muscle as it takes a lighter shade of 

 staining. 



Note: Azophloxine is used here as a substitute for ponceau de 

 xylidine in Goldner's modification of Masson's technique. 



The stain is also suggested in place of Eosin as a counterstain 

 for use with haematoxylin. The advantages of using azophloxine 

 are that it gives clear and delicate pictures and it does not overstain, 

 if the recommended procedure is followed. When azophloxine 

 is to be used merely as a counterstain for haematoxylin the pro- 

 cedure is as follows : 



I. Proceed as steps, i, 2, 3, 4, and 5 (above). 

 II. Rinse in distilled water. 



III. Dehydrate with three changes of absolute alcohol; then 

 clear and mount. 



Reference: Halper, M. H. (1954, November), Stain Tech., 29, no. 6, 315-7. 



Ill 



