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Perls’ Prussian Blue for Hemosiderin

Perls' Prussian Blue

for Hemosiderin

7
steps
4
materials

Materials

  • Neutral red
  • Stock solution A
    MaterialAmount
    Potassium ferrocyanide2g
    Distilled water100mL
  • Stock solution B
    MaterialAmount
    Hydrochloric acid, conc.2mL
    Distilled water100mL
  • Working solution
    MaterialAmount
    Stock solution A1volume
    Stock solution B1volume

Make the working solution immediately before use. Do not use if more than 30 minutes old.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Avoid iron containing materials and jars while fixing as these may contaminate the tissue. Acid containing fixatives may remove some of the iron deposits, but apart from that most are satisfactory.

Protocol

  1. Bring sections to distilled water with xylene and ethanol.
  2. Place into the working solution for 15 minutes.
  3. Rinse with distilled water, then tap water.
  4. Stain with neutral red for one minute.
  5. Rinse well with tap water.
  6. Dehydrate with ethanol.
  7. Clear with xylene.

Expected Results

  • Ferric iron  –  blue
  • Nuclei  –  red

Notes

  • Avoid washing with tap water before placing into the working solution, as rust in the water or tap fixtures could cause false positive staining.
  • Wash well at step 3, as traces of iron will form a granular red deposit with neutral red.
  • Iron ores can be demonstrated, but the acid concentration in solution A may need to be increased to 10% or more.
  • Hemosiderin forms the body’s iron stores. The iron is in the ferric state, and may be demonstrated by releasing it from hemosiderin with hydrochloric acid, forming ferric chloride. The iron reacts with potassium ferrocyanide
    to form ferric ferrocyanide. This is an insoluble, blue compound known as Prussian blue or Berlin blue. The intensity of the colour gives some indication as to amount, but it is qualitative only. Other sources of ferric iron will also be demonstrated.
  • The reaction is: 4FeCl3 + 3K4Fe(CN)6 = Fe4[Fe(CN)6]3 + 12KCl

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Culling, C.F.A., Allison, R.T. and Barr, W.T.
    Cellular Pathology Technique, Ed.4.
    Butterworth, London, UK.
  2. Susan Budavari, Editor, (1996)
    The Merck Index, Ed. 12
    Merck & Co., Inc., Whitehouse Station, NJ, USA