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Calcium Staining in Histology

There are many stains out there to histologically stain for calcium but we use primarily 2.  They both give different color results and use different chemicals.  The first is called Alizarin Red S.  This stain can be visualized with or without a polarizing microscope.  The polarized microscope makes the calcium deposits much brighter (see picture below).  As the picture suggests, we also carry positive control slides for this and other calcium stains.  We like this one the best because it is a much quicker stain that can be completed in 25 minutes using paraffin slides.  The chemicals necessary for the stain are alizarin Red S powder and ammonium hydroxide.  You also need a pH meter to get the solution to a pH range of 4.1-4.3.

The other stain is Von Kossa‘s method for calcium.  This is a much longer stain (1 hour 41 minutes) that uses silver nitrate to turn the calcium salts black.  To achieve good results with this stain, the tissue sections must be exposed to direct sunlight, a UV lamp or a 100 watt lamp light while immersed in the silver nitrate.  In our opinion this stain is more finicky and easier to foul up than the Alizarin Red stain.  Unlike most silver protocols, the glassware in this protocol will not turn black.  The Von Kossa solution protocol will be posted on the protocol solutions the day after today.

Human Kidney  Von Kossa
Human Kidney Von Kossa



How to clean glassware after doing silver stains:

After the stain is complete and the controls tissues have been verified, the toning solution (gold chloride) can be used to clean all glassware.  Keep in mind that this solution is very toxic and should be disposed of properly (not down the sink).


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Special Stain Solution Protocols Page

Hello,  We have starting adding our solution protocols on a new page.  These are all the protocols that we use now. A professional histology service.

An Expert Histology Service

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Alizarin Red S Stain Protocol for Calcium

Run a control slide. Control tissue: Bone or tissue with calcium present

Safety equipment: Work under a hood with lab coat, gloves, and safety glasses.

  1.   De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
  2.   De-paraffin slides in xylene (2)——————- 2 minutes (re-use)
  3.   Clear slides in 100% alcohol———————- 2 minutes
  4.   Clear slides in 100% alcohol———————- 2 minutes
  5.   Hydrate slides in 95% alcohol——————– 2 minutes
  6.   Hydrate slides in 50% alcohol——————– 1 minute
  7.   Rinse rapidly in DH2O—————————— 30 seconds
  8.   Cover the sections with alizarin Red S solution (re-use)
  9.   Observe the reaction under a microscope and remove when the red-orange lake forms ( 5 minutes to 15 mintues). The lake should be heavy but not too diffuse.

10.    Shake off excess dye and blot carefully.

11.    Dehydrate in acetone for 10 to 20 seconds (dump)

12.    Dehydrate in acetone-xylene (50:50)———–10 to 20 seconds (dump)

13.    Clear in xylene—————————————- 5 minutes (re-use)

14.    Coverslip


Calcium Deposits (except oxalate) ———————Orange-Red

This precipitate is birefringent.


An Expert Histology Service

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Special stains protocols

I am trying to figure out what to do about the special stains protocols.  There are about 35 of them.  Putting them on the front page does not seem to be efficient.   I would like them all to be searchable and downloadable, maybe on a different page.  What do you think, does it make a difference?