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 suspension 1 vol
Acetic acid, 2M 1 vol
Perls' solution
2% potassium ferrocyanide 1 vol
2% hydrochloric acid 1 vol

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be 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


  1. 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.
  2. 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.
  3. 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.
  4. A PAS may be applied following step 7, in which case the colour 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.
  5. 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.


Culling C.F.A., (1963)
Handbook of histopathological and histochemical techniques Ed. 2
Butterworth, London, UK.

Bancroft, J.D. and Stevens A. (1982)
Theory and practice of histological techniques Ed. 2
Churchill Livingstone, Edinburgh & London, UK.

Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.




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