Re: [Histonet] Yasue's Silver Nitrate-Rubeanic Acid Method for Calcium Oxalate

From:Sharon Bledsoe


Our Lab has been using the Yasue Stain for years now.

Yasue Method of Staining Calcium
Histochemical Identification of Calcium Oxalate
Acta Histochem, Cytochem, Vol 2(3), 1969, pp 83-95.

       This is a metal substitution method for calcium histochemistry. 
Metal substitution depends on a reaction to substitute calcium, which 
is bound to anion, with other heavy metals, and therefore it does not 
detect calcium cation directly but such anions as carbonate, 
phosphate, sulfate and oxalate.
       Rubeanic acid reacts with silver and some other metals to form 
a colored double salt.  A brownish-black insoluble double salt is 
produced with a reaction of rubeanic acid and silver, the reaction is 
very sensitive and specific if there are no other salts.  Slightly 
deposited silver particles on the surface of crystals of calcium 
oxalate with a low ability to react are assumed to be blackened with 
rubeanic acid and its sensitivity as well as localization are good 
enough to demonstrate calcium oxalate.
       This reaction can not be taken as a specific one for calcium 
oxalate. Calcium Oxalate crystals are demonstrated histochemically 
when optical characteristics (birefringence), hematoxylin and eosin 
staining (lack of), solubility test (soluble in 2% HCl, not soluble 
in 5% acetic acid), microincineration method and gypsum formation 
reaction are combined.


1.  Removal of Calcium Phosphate and Calcium Carbonate
soak in 5% Acetic Acid for 30 minutes.

2.  Rinse in running distilled water for 3 minutes

3.  Immerse in 5% Aqueous Silver Nitrate (AgNO3)
	10 - 20 minutes. 
(12 minutes seemed to work best for the rat kidneys--15 minutes left 
a film on top of the tissue that was probably from the Rubeanic Acid 
in the next step).

4.  Rinse in running distilled water for 3 minutes

5.  Add 2 drops of strong Ammonia Water (10 %) to a Coplin jar filled 
with saturated
Rubeanic Acid.
Immerse slides for about 1 minute.

6.  Rinse in 50 % EtOH followed by distilled water.

7.  Counterstain with Methyl Green or Safranin O.

8.  Dehydrate, clear and mount

Calcium deposits are colored dark brown or black.


	5% AgNO3     
		(5 gm / 100 ml of ddH2O)

	Saturated Rubenic Acid
		About 300 mg / 100 ml of 70% EtOH

	Safranin O:
		Heat water to about 80 degrees, add  about 4 gm / 100 ml.
		Stain is very strong.. needed to dilute.. took about 
2 seconds to stain.

We have some nice images of the staining in the following:

Evan, AP et al. 2003, Randall's plaque of patients with 
nephrolithiasis begins in basement membranes of thin loops of Henle. 
Journal of Clinical Investigation.111:607-616.

Evan, AP et al. 2005.  Crystal-associated nephropathy in patients 
with brushite nephrolithiasis. Kidney International 67:576-591

Evan, AP et al. 2005. Apatite plaque particles in inner medulla of 
kidneys of calcium oxalate stone formers: Osteopontin localization. 
Kidney International 68:145-154

Hope this helps,

Sharon Bledsoe
Research Analyst
Indiana University, School of Medicine
Department of Anatomy & Cell Biology
MS 5055P, 635 Barnhill Drive
Indianapolis, IN 46202


At 6:01 PM -0400 8/1/07, wrote:
>Does anyone have the protocol for Yasue's Silver Nitrate Rubeanic 
>Acid method. One of the researchers is doing a calcium oxalate 
>study. I have already given him Pizzolato's, Von Kossa, and Alizarin 
>Red for comparisons. I have heard that the Yasue's method is much 
>more specific.
>Thanks, Donna
>Donna Emge, HT(ASCP)
>Northwestern University
>Feinburg School of Medicine
>303 E Superior St. Lurie 7-220
>Chicago, IL 60611
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