346 Histochemistry (chap. 21) 



Trisbuffer, pH 7.19: 



0.2 M tris (hydroxymethyl) aminomethane .... 25.0 ml. 



O.IN HCl 45.0 ml. 



dilute with distilled water to 100.0 ml. 



Working solution: 



stock substrate 1.0 ml. 



distilled water 40.0 ml. 



tris buffer 10.0 ml. 



Garnet GBC (diazotized o-aminoazotoluene) . . 30.0 mg. 



Shake and filter. 



For substrate, the alanyl compound may be substituted: 10 mg. in 40 

 ml. hot distilled water (90°C). Cool and add 10 ml. of tris buffer and 

 30 mg. garnet GBC. 



procedure: 



1. Chloroform or petroleum ether, 2 changes: 4 minutes each. 



2. Absolute acetone: 1 minute. 



3. 95% acetone: 1 minute. 



4. 85% acetone, several dips. 



5. Distilled water, few dips. 



6. Substrate: 15 minutes to 1 hour, room temperature. 



7. Tap water: 5 minutes. 



8. Hematoxylin: 3 minutes, 



9. Tap water: 10 minutes. 

 10. Glycerol jelly. 



results: 

 sites — red 



comments: 



If necessary to purify GBC salt (not Imperial Chemical Industries 

 product): 



1. Dissolve 10 gms. salt in 50 ml. methyl alcohol. Let stand 0.5 hour. 

 Filter. 



2. Repeat with residue. 



3. Dissolve second residue in 500 ml. ether. Let stand 2-3 hours. 



4. Filter. Wash residue 2-3 times with ether. Store dry powder in re- 

 frigerator. 



"Imperial Chemical Industries, Manchester, England, recommended. 



