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Alizarin Red S Staining Protocol for Calcium
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Alizarin Red S Staining Protocol for Calcium

Description: Alizarin Red S, an anthraquinone derivative, may be used to identify calcium in tissue sections. The reaction is not strictly specific for calcium, since magnesium, manganese, barium, strontium, and iron may interfere, but these elements usually do not occur in sufficient concentration to interfere with the staining. Calcium forms an Alizarin Red S-calcium complex in a chelation process, and the end product is birefringent.

Fixation: Neutral buffered formalin or alcoholic formalin fixed, paraffin embedded tissue sections.

Positive Control: mouse embryo or a know calcium containing tissue sections

Solution and Reagents:

Alizarin Red Solution:

    Alizarin Red S (C.I. 58005) ----------- 2 g

    Distilled water ------------------------- 100 ml

    Mix well. Adjust the pH to 4.1~4.3 with 10% ammonium hydroxide. The pH is critical, so make fresh or check pH if the solution is more than one month old.

Acetone (100%)


   Acetone (100%) ------------------------- 50 ml

   Xylene ----------------------------------- 50 ml


  1. De-paraffinize slides to distilled water.
  2. Stain slides with the Alizarin Red Solution for 30 seconds to 5 minutes, and observe the reaction microscopically. Usually 2 minutes will produce nice red-orange staining of calcium.
  3. Shake off excess dye and blot sections.
  4. Dehydrate in acetone, 20 dips. Then in Acetone-Xylene (1:1) solution, 20 dips.
  5. Clear in xylene and mount in a synthetic mounting medium.


Calcium deposits (except oxalate) -------- orange-red

This precipitate is birefringent.

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