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Hale’s Colloidal Iron for Acid Mucosubstances

Hale's Colloidal Iron

for Acid Mucosubstances


Using the colloidal iron suspension of Rhinehart and Abu’l Haj.



  • Neutral red
  • Acetic acid, 2M (12%)
  • Potassium ferrocyanide, 2% aqueous
  • Hydrochloric acid, 2% aqueous
  • Rhinehart & Abu’l Haj’s colloidal iron suspension

Working colloidal iron

Colloidal iron suspension1vol
Acetic acid, 2M1vol

Perls’ solution

2% potassium ferrocyanide1vol
2% hydrochloric acid1vol

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are usually satisfactory.


  1. Bring sections to water via xylene and ethanol.
  2. Place into the working colloidal iron for 15-20 minutes.
  3. Wash with distilled water.
  4. Wash with running tap water for 5 minutes to remove all traces of colloidal iron
  5. Wash with distilled water.
  6. Place into freshly made Perls’ solution for 10 minutes.
  7. Wash with distilled water.
  8. Counterstain with neutral red for 1 minute.
  9. Dehydrate with ethanols.
  10. Clear with xylene and mount with a resinous medium.

Expected Results

  • Acid mucopolysaccharides  –  blue
  • Nuclei  –  red


  • The original method used a commercial colloidal iron preparation. This is still available. However, the colloidal iron suspension of Rhinehart and Abu’l Haj is reputed to produce a cleaner background. Other colloidal iron suspensions have also been recommended.
  • Since this method depends on the staining of iron compounds with the prussian blue reaction, any hemosiderin present will also be stained. If this is a concern, a control section should be stained which has not been treated with colloidal iron. Material stained blue in both sections should be discounted.
  • Nuclear fast red may also be used as a nuclear counterstain, or a Feulgen’s nucleal reaction may be applied before step 2, in which case the nuclear counterstain should be omitted.
  • A PAS may be applied following step 7, in which case the color of the nuclear counterstain should be changed, perhaps with a strictly progressive hemalum. Acid mucosubstances will be stained blue in contrast to red neutral mucosubstances. However, they are often present as mixtures and the contrast may not be clear.
  • Longley’s variant of this method includes a Feulgen’s nucleal reaction before step 2, and a Wiegert van Gieson counterstain following step 7, so that nuclei are black, cytoplasm is yellow and collagen red.

Safety Note

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


  1. Culling C.F.A., (1963)
    Handbook of histopathological and histochemical techniques Ed. 2
    Butterworth, London, UK.
  2. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
  3. Lillie, R.D., (1954)
    Histopathologic technique and practical histochemistry Ed.2
    Blakiston, New York, USA.