Apathy's
Alum Hematoxylin

Ingredient	Amount	Function
Hematoxylin	3 g	Dye
Ammonium alum	30 g	Mordant
Distilled water	450 mL	Solvent
95% ethanol	250 mL	Dye solvent
Glycerol	350 mL	Stabiliser
Glacial acetic acid	10 mL	Acidifier
Salicylic acid	0.3 g	Acidifier

 

Compounding procedure
Dissolve the dye in 100 mL water and 250 mL ethanol.
Leave at room temperature to ripen (months).
Dissolve the alum and acids in 350 mL water.
Combine both solutions.
Add glycerol.

Method

1. Bring sections to water with xylene and ethanol.
2. Place into the staining solution for an appropriate time.
3. Rinse well with water.
4. Differentiate with acid ethanol if necessary.
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. Lillie excludes the salicylic acid.
2. The appropriate staining time should be determined by trial.
3. Acid ethanol is 0.5% - 1% hydrochloric acid in 70% ethanol.
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
Gray, Peter. (1954)
The Microtomist's Formulary and Guide.
Originally published by:– The Blakiston Co.
Republished by:– Robert E. Krieger Publishing Co.

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