Perls’ Prussian blue reaction method is used for the detection of iron in tissues.

This method mostly stains iron in the state of ferritin and haemosiderin.


Potassium ferrocyanide in acid solution (diluted hydrochloric acid) unmask ferric iron in the form of the hydroxide {Fe(OH)3}.

The ferric iron in the solution reacts with diluted potassium ferrocyanide solution to produce Prussian blue colour insoluble compound known as ferric ferrocyanide.


Equal volumes of 1% aqueous potassium ferrocyanide and 2% aqueous hydrochloric acid are the solutions necessary for this method.

Equal volumes of above solutions should be mixed freshly before starting the staining procedure.

  • First bring the control slide and the test slide down to the water through a series of alcohol solutions.
  • Then cover the sections with freshly mixed acid ferrocyanide solution.
  • Stain for 10-30 minutes. Next wash well with distilled water.
  • Then stain with 0.2% aqueous safranin solution.
  • Then wash rapidly with water. Dehydrate, clear and mount with DPX.


Iron stains in Prussian blue colour.

Nuclei stain in red colour and background stains in pale red colour.

Special Consideration

When in the fixation step of the tissues, it should be avoided using acid fixatives.

Because hemosiderin soluble in acid solutions.

It may alter the perls’ reaction and results may be false negative.

Chromates in the solutions can also be interfered with the preservation of iron.

The staining time may be vary depending on the amount of ferric iron present in the solution.

Instead of aqueous safranin solution, 0.5% aqueous neutral red, 0.1% nuclear fast red or eosin can be used.

In unfixed tissues, haemosiderin is soluble in acid solutions and insoluble in alkaline solutions.

However, after fixation with formalin, it is slowly soluble in diluted acids such as oxalic acid.

Some irons such as haemoglobin and myoglobin found in tissues cannot be demonstrated by traditional methods.

Those irons are tightly bound within protein complexes.

These types of irons can be released or unmasked by treating with hydrogen peroxide.

Similarly acid ferrocyanide solution can be heated at 60°C in a water bath to release iron from the tissues which are bound with protein complexes.

Because of this heating, sometimes a fine, diffuse, blue precipitate can be occurred on both the tissue section and slide, this can be prevented by staining slides at room temperature.

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