Horneyold's
Alum Hematoxylin

Hematoxylin solution	Amount	Function
Hematoxylin	0.7 g	Dye
100% ethanol	20 mL	Solvent
Alum solution	Amount	Function
Ammonium alum	0.35 g	Mordant
Distilled water	60 mL	Solvent
Tincture of iodine	Amount	Function
Iodine	70 g	Oxidant
Potassium iodide	50 g	–
Distilled water	50 mL	Solvent
100% ethanol	950 mL	Solvent

 

Compounding procedure

Tincture of iodine
Mix the dry iodine and potassium iodide together.
Add the water and mix well to dissolve.
Add the ethanol and mix well.

Staining solution
Dissolve the hematoxylin in the ethanol and the alum in the water.
Combine the two solutions and mix well.
Leave in the light by a window for 3-4 days.
Add 20 drops tincture of iodine.

Method

1. Bring sections to water with xylene and ethanol.
2. Place into the staining solution for 5-10 minutes.
3. Rinse well with water.
4. Differentiate with acetic ethanol.
5. Rinse with water and blue.
6. Rinse well with water.
7. Counterstain if desired.
8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

• Nuclei  –  blue
• Background  –  as counterstain or unstained

Notes

1. Horneyold's formula is a modification of Böhmer's alum hematoxylin.
2. This solution is regressive, and is recommended for osmium fixed tissue.
3. Acetic ethanol is 70% ethanol containing glacial acetic acid. The amount of acetic acid to add was not given, but 0.5%-1% should suffice.
4. Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

 

Reference
Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
The Microtomist's Vade-Mecum. 11 ed.,
Churchill, London, UK.

Lange, N.A., (1966)
Lange's Handbook of Chemistry, Revised 10th ed.,
McGraw-Hill.