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Azure A Staining Protocol for Mast Cells
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Azure A Staining Protocol for Mast Cells

Prepared by


IMVS Division of Pathology

The Queen Elizabeth Hospital

Woodville Road, Woodville, South Australia 5011


Mast cells were originally described by von Recklinghausen in the early 1870s.  Mast cells are characterised by cytoplasmic granules which are rich in heparans (highly sulphated proteoglycans), which are demonstrated metachromatically by the thiazin group of dyes.

Metachromasia - (one dye exhibiting two different colour reactions) Although not fully understood, metachromasia is thought to be an optical phenomenon produced by the polymerisaton of the dye molecules at the chemical site of binding.  Three forms may be identified:

  • a-metachromasia - monomeric form, usually blue
  • b-metachromasia - loose complex form, usually blue/purple
  • g-metachromasia - dimeric form, usually red

Technical difficulties are encountered during the dehydration stages of staining, as the metachromatic reaction is easily lost.  Controversy exists as to whether true metachromacy requires the definition to include aqueous mounting media.

The ability to react metachromatically is a property exhibited by certain negatively charged entities (polyanions) and certain cationic dyes. These react to form dye polymers which have a different absorption characteristic to the normal form of the dye (monomer) and therefore a different colour emission. Azure A exhibits a blue orthochromasia and a purple-red metachromasia. The intensity of the metachromasia appears to be conferred by the initial oxidation step in potassium permanganate which tends to aid cationic dye uptake by the tissue, generally. The alcohol fastness and increased brightness of metachromasia is attributed to the use of zinc sulphate which is a mordant type differentiator.

Technical Points

Poor staining can usually be traced back to a faulty batch of dye.


1.      Bring sections to distilled water.

2.      Oxidise in 1% aqueous potassium permanganate 5 mins

3.      Rinse distilled water

4.      Decolourise with 2% aqueous oxalic acid for 1 min

5.      Wash in running water for 3mins

6.      Rinse with distilled water

7.      Stain in azure A 5 mins

8.      Differentiate in 1% zinc sulphate until the section is macroscopically pale blue.

9.      Rinse in distilled water.

10.    Blot, and allow to completely dry.

11.    Clear in xylene and mount.


Mast cell granules, sulphated and carboxylated mucins --------- purple

Nuclei -----------------------------------------------------------------  blue

Background ----------------------------------------------------------- pale blue

Reagent Formulae

1.      1% aq potassium permanganate

2.      2% oxalic acid

3.      0.1% azure A in 30% ethanol
       azure A (CI 52005) ------- 0.1 g
       distilled water ------------ 70.0 mls
       ethanol -------------------- 30.0 mls
Dissolve the dye in the distilled water.  Add the alcohol, and mix well.  Filter before use.  The stain keeps well for 2 years.

4.   1% zinc sulphate
            zinc sulphate (ZnSO4.7H20) Analar --- 0.1 g
            distilled water -------------------------- 100.0 ml


Hughesdon PE,(1949),J.R.microsc.Soc.,69,1

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