Smith's Zirconyl Hematoxylin
McNulty's Modification

This hematoxylin substitutes for alcian blue pH 2.5 for staining acid mucins.

Ingredient	Amount	Function
Hematoxylin	100 mg	Dye
Ethanol, absolute	5 mL	Solvent
Sodium iodate, 0.5% aqu.	5 mL	Oxidant
Zirconyl chloride, octahydrate	400 mg	Mordant
Distilled Water	22.5 mL	Solvent
Glycerol	7.5 mL	Anti-oxidant

 

Compounding procedure
Dissolve the hematoxylin in the ethanol.
Add the sodium iodate solution (freshly made).
Add the zirconyl chloride.
Combine the glycerol and water, and add to the solution.
Stir 5 minutes.

Method

1. Bring sections to water with xylene and ethanol.
2. Place into the staining solution for 10 minutes.
3. Wash with distilled water, two changes of 2 minutes each.
4. Counterstain in methylene green for 5 minutes.
5. Wash with distilled water, two changes of 2 minutes each.
6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  green
• Acid mucins  –  Purple

Notes

1. Formalin fixed, paraffin sections are suitable.
2. The counterstain recommended was 0.05% methylene green in 2.4% boric acid.
3. This method was recommended as a substitute for alcian blue staining of acid mucins (pH 2.5).
4. Staining may be removed with 1% hydrochloric acid in 70% ethanol.
5. The solution above is McNulty's modification of Smith's original, and stains more intensely:
   • Hematoxylin, 100 mg
   • Absolute ethanol, 5 mL
   • 0.1% aqueous sodium iodate, 5 mL
   • Zirconyl chloride octahydrate, 400 mg
   • 22% aqueous glycerol, 45 mL
   • Glacial acetic acid, 5 mL

 

Reference
Smith, A. A., (1999)
Zirconyl hematoxylin staining of acidic mucins.
Journal of Histochemistry and Cytochemistry, v. 47, p. 1645.

McNulty, J. M. and Smith, A. A., (2004)
An improved formulation of the zirconyl hematoxylin stain for acidic mucins.
Biotechnic and Histochemistry, v. 79, Nº 5, p. 191.

McNulty, J. M., Kambour, M. J. and Smith, A. A., (2004)
Use of an improved zirconyl hematoxylin stain in the diagnosis of Barrett's esophagus.
J. Cell. Mol. Med., v. 8, Nº 3, p. 382.