Pusey's
Alum Hematoxylin

This solution is described as a modification of Mayer's hemalum, but please refer to that formula.

Ingredient	Amount	Function
Hematoxylin, aged 10% ethanolic	16 mL	Dye
Ammonium alum	60 g	Mordant
Distilled water	1 L	Solvent
Sodium iodate	0.25 g	Oxidant
Citric acid, 5% aqueous	7 mL	Acidifier
Chloral hydrate	50 g	Stabiliser

 

Compounding procedure
Dissolve the alum in the water using low heat, but do not boil.
Add the hematoxylin solution and mix well.
Add the sodium iodate and dissolve.
Leave for 30 minutes.
Add the chloral hydrate and dissolve.
Add the citric acid and mix well.
Check that the pH is 2.45, adjust with citric acid if not.
It may be used immediately.

Method

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

Expected results

• Nuclei  –  blue
• Background  –  as counterstain or unstained

Notes

1. The alcoholic hematoxylin solution should be aged (partially oxidised).
2. The staining time was not given, but 5-10 minutes should suffice.
3. 16 mL of a 10% solution of hematoxylin equates to 1.6 grams dye. With 60 grams of mordant and strong oxidation this would indicate a darkly staining progressive solution.
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
Pusey's modified Mayer's hematoxylin,
Journal of Histotechnology, v.2, No.2, p.54, 1979
citing:
  Villaneuva, A.R., (1976)
  Methods of preparing and interpreting mineralized sections of bone, in:
  Proceedings of the First Workshop on Bone Morphometry
  Jaworski, Z. F. G., Editor, (1976).