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
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 gm / 100 ml of ddH2O)
Saturated Rubenic Acid
About 300 mg / 100 ml of 70% EtOH
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,
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, email@example.com 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
>Donna Emge, HT(ASCP)
>Feinburg School of Medicine
>303 E Superior St. Lurie 7-220
>Chicago, IL 60611
>AOL now offers free email to everyone. Find out more about what's
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