Teslirifr for Cnlcium 237 



solutions: 



Silver nitrate: 



silver nitrate, AgNOs 5.0 gm. 



distilled water 100.0 ml. 



Fresh solution always best; never use one more than 1 week old. 

 Safranin, see page 233. 



procedure: 



1. Deparaffinize and hydrate slides to distilled water; remove HgClo. 



2. Treat with silver nitrate in dark: 30 minutes. 



3. Rinse thoroughly in distilled water. 



4. Expose slides (in distilled water) to bright light (75-lOOW bulb 

 satisfactory): 1 hour. Lay them o\er a white backgroimd to expe- 

 dite reaction. 



5. Wash thoroughly in distilled water. 



6. Counterstain in safranin (or other red nuclear stain): 2-3 minutes. 



7. Rinse in 70% alcohol. 



8. Dehydrate, clear and mount. 



results: 



calcium deposits — dark brown to black 



nuclei and other tissue elements — shades of red and rose 



comments: 



If iron blocks out the calcium, treat 10-15 minutes with .005M so- 

 dium EDTA (ethylenediaminetetraacetic acid) in normal saline. 

 Wash in distilled water and proceed to step 2. {McGee-Russell, 19'^S) 



This reaction actually demonstrates phosphates and carbonates 

 rather than calcium itself, but since soluble phosphates and carbon- 

 ates are washed out, the calcium phosphates and calcium carbonates 

 remain to react with the silver (Lillie, 19343) and the test can be 

 regarded as sufficiently specific (Pearse, 1953). 



Renaud (1959) demonstrates that a high percentage of alcohol (at 

 least 80% content) in the fixing fluid is necessary in order to detect 

 calcium in some tissues (i.e., heart and coronary vessels). Water and 

 even buffered formalin can dissolve out some small deposits of cal- 

 cium salts. 



Alizarine Red S, pearse (1953) 



fixation: preferably one containing 80% alcohol, for maximiun preser- 

 vation of calcium. 



