FIXATION:        95% ethyl alcohol or 10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

1% Alizarin Red S Solution

Alizarin red S, C.I. 58005 ------------------------------------------------------                1.0 gm

Distilled water ------------------------------------------------------------------                100.0 ml

       Stir dye into the distilled water so that only a few small grains of dye

       remain undissolved.  Add 10% ammonium hydroxide slowly with

       constant stirring until the pH is 6.3-6.5.  Solution is stable for one year.

10% Ammonium Hydroxide Solution

Ammonium hydroxide, 28% -----------------------------------------------------        5.0 ml

Distilled water -------------------------------------------------------------------        45.0 ml

0.2% Fast Green F.C.F. Solution (stock)

Fast green F.C.F., C.I. 42053 --------------------------------------------------                0.2 gm

Distilled water ------------------------------------------------------------------                100.0 ml

Acetic acid ---------------------------------------------------------------------                0.2 ml

Fast Green Solution (working)

Fast green (stock) --------------------------------------------------------------                10.0 ml

Distilled water ------------------------------------------------------------------                35.0 ml

• Use positive control slide.

1. Deparaffinize and hydrate to 95% alcohol.
2. Drain off excess alcohol and allow the sections to dry.
3. Place in alizarin red S solution for 5 minutes.
4. Remove excess stain with distilled water five or six rinses.
5. Counterstain in fast green working solution for 40 seconds.
6. Rinse in three changes of distilled water.
7. Dehydrate in graded alcohols.
8. Clear in xylene, three or four changes.
9. Mount with synthetic resin.

Calcium salts ------------------------------------------------------                    intense reddish orange

Background -------------------------------------------------------        pale green

Some calcium salts are soluble in formalin fixatives.  Fixation time should be brief, or ethanol can be used.  This method may be slightly more sensitive than the von Kossa stain for calcium.

Some histochemistry textbooks indicate that the alizarin red S solution, used in this method, be adjusted to pH 4.2.  We found that calcium will stain equelly well at this pH as it does at pH 6.3-6.5.

Sections must not be hydrated beyond 95% alcohol after being deparaffinized.  If the sections are taken to distilled water any calcium salts which may be present will be removed.

Some samples of alizarin red S may cause the staining solution to become a semiliquid gel.  If this occurs shake the solution well before use.  The solution will become more liquid-like and produce satisfactory staining results.

Dahl, L.K.:  A simple and sensitive histochemical method for calcium.  J. Exp. Med. 95:474-479, 1952.

Luna, L.G.:  Histopathologic Methods and Color Atlas of Special Stains and Tissue Artifacts, Maryland, American Histolabs, One., Publications Division, 1992, pp. 327-328.