Perl’s prussian blue staining technique for hemosiderin

Hemosiderin is present in tissues as intracellular pigment. It contains iron in the form of ferric hydroxide that is bond to a protein framework.

Principle

The reaction occurs with the treatment of tissue sections with acid ferrocyanide solution. Any ferric ion (Fe3+) in the tissue combines with ferrocyanide and results in the formaion of a bright blue pigment called “prussian blue” or ferric ferrocyanide.

Fixation

Avoid the use of acid fixatives. Chromates will also interfare with the preservation of iron.

Staining solution

• 1% aqueous potassium ferrocyanide = 20 ml
• 2% aqueous hydrochloric acid = 20 ml
• Mix both

Procedure

1. Deparaffinize and bring the sections to water.
2. Treat the sections with freshly prepared acid ferrocyanide solution for 10-30 minutes.
3. Wash well in distilled water.
4. Lightly stain the nuclei with 0.5% aqueous neutral red or 0.1% nuclear fast red.
5. Wash rapidly in distilled water.
6. Dehydrate, clear and mount.

Result and Interpretation

• Ferric ion = blue
• Nuclei = red
• Background = pink