Goldman's
Iron Hematoxylin

Solution A	Amount	Function
Ferric ammonium sulphate	4 g	Mordant
Distilled water	100 mL	Solvent
Acetic acid, glacial	1 mL	Acidifier
Sulphuric acid	0.12 mL	Acidifier
Solution B	Amount	Function
Picric acid, saturated aqueous	100 mL	Dye and acid
Sulphuric acid	0.1 mL	Acidifier
Solution C	Amount	Function
Hematoxylin	0.5 g	Dye
Distilled water	100 mL	Solvent
Solution D	Amount	Function
70% ethanol	100 mL	Solvent
Lithium carbonate, saturated aqueous	5 drops	Base

 

Compounding procedure
Make each solution seperately.
The aqueous hematoxylin should be ripened before use.

Method

1. Bring sections to water, removing mercury pigment if necessary.
2. Place into solution A for 30 minutes - 24 hours.
3. Wash in running tap water for 10 minutes.
4. Place in solution B for 3 hours or longer.
5. Wash in running tap water for 15 minutes.
6. Place in solution C for 1 hour.
7. Wash in running tap water for 15 minutes.
8. Blue with solution D.
9. Counterstain if desired.
10. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  black
• Basophil cytoplasm  –  grey
• Background  –  as counterstain or unstained

Notes

1. The stock solutions are stable for some time.
2. The technique was originally intended for the demonstration of protozoa.
3. The method was designed for paraffin sections of material fixed with formalin variants, Bouin's or Zenker's fluids.
4. Overstaining occurs only if sections are left in hematoxylin for several hours.
5. Bouin fixed tissue is not as intensely stained as with other fixatives.
6. The time in picric acid (solution B) is necessary, reducing it causes overstaining.

 

Reference
Lillie, R.D., (1954)
Histopathologic technique and practical histochemistry Ed.2
Blakiston, New York, USA.
  Citing:–
    Goldman, (1951)
    American Journal of Pathology, v.21, p.198