238 Pigmenfs and Minerals (chap. 17) 



solutions: 



Alizarine red 5; 



alizarine red 5, C.l. 58005 1.0 gm. 



distilled water 100.0 ml. 



Toliiidine blue: 



toluidine blue O, C.I. 52040 0.1 gm. 



distilled water 100.0 ml. 



procedure: 



1. Deparaffinize and hydrate slides to water. 



2. Stain in alizarine red: 3-5 minutes. 



3. Rinse in distilled water. 



4. Stain in toluidine blue: 1 minute. 



5. Rinse in distilled water. 



6. Dehydrate, clear and mount. 



results: 



calcium — orange-red 



nuclei — blue 



inorganic iron (not hemosiderin) — purple 



comments: 



Any counterstain should be kept light to provide contrast for the 

 alizarine red color. 



Hemoglobin Staining 



Benzidine Method (ralph's, 1941; click, 1949) 

 fixation: alcohol or formalin. 



solutions: 



Benzidine reagent: 



benzidine 1.0 gm. 



absolute methyl alcohol 100.0 ml. 



Peroxide reagent: 



peroxide (reagent 3%) 25.0 ml. 



70% ethyl alcohol 75.0 ml. 



procedure: 



1. Deparaffinize sections and hydrate to water. Not necessary for 

 smears, which will be fixed by benzidine reagent. 



2. Flood with benzidine reagent: 1-2 minutes. 



