Smith's Vanadium Hematoxylin
for Basic Proteins including Histones

When differentiated with dilute lithium carbonate this solution demonstrates basic proteins, including nuclear histones.

Ingredient	Amount	Function
Hematoxylin	50 mg	Dye
Ethanol, 100%	5 mL	Solvent
Glycerol	10 mL
Distilled water	35 mL	Solvent
Ammonium meta-vanadate	200 mg	Solvent

 

Compounding procedure
Dissolve the hematoxylin in the ethanol, then add the glycerol and water in sequence.
Add the ammonium vanadate and stir for 30 minutes.
The ammonium vanadate may not completely dissolve.
The pH is 6.2

Tissue
Paraffin sections of Schaudinn, Bouin or formalin fixed tissues are suitable. Sections of Zenker or Helly fixed tissues must be soaked in saturated aqueous lithium carbonate for 4 hours to stain satisfactorily.

Method

1. Bring sections to water with xylene and ethanol.
2. Place into the vanadate hematoxylin for 30 minutes.
3. Place directly into 0.08% aqueous lithium carbonate for the appropriate time.
4. Briefly rinse with distilled water for 2 seconds.
5. Optionally, counterstain if wished.
6. Dehydrate with 70%, 95% and absolute ethanols.
7. Clear with xylene and mount with a resinous medium (Permount specified).

Expected results

• Nuclear histone  –  blue
• Ribosomal protein  –  paler blue
• Other protein  –  as counterstain or unstained

Differentiation times in lithium carbonate
Bouin	2 minutes
Formalin, 10%	2 minutes
Helly	10 minutes
Schaudinn	5 minutes
Zenker	10 minutes

 

Notes

1. Counterstaining can be accomplished at step 5 with 1% aqueous eosin Y, phloxine B or erythrosin B.
2. Alternatively, metanil yellow at 0.1% in 95% ethanol containing 0.1% acetic acid may be used between the 70% and 95% ethanols in step 6.
3. Counterstaining with 0.01% aqueous safranin O stained RNA intensely, giving good contrast between blue nuclear histone and endoplasmic reticulum.

 

Reference
Smith, A. A., (1995)
A vanadate hematoxylin stain for basic proteins.
Biotechnic and Histochemistry, v. 70, Nº 5, p. 5.