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Horneyold’s Alum Hematoxylin

Horneyold's Alum Hematoxylin

8
steps
6
materials

Materials

Hematoxylin Solution

MaterialAmountFunction
Hematoxylin0.7 gDye
100% ethanol20 mLSolvent

Alum Solution

MaterialAmountFunction
Ammonium alum0.35 gMordant
Distilled water60 mLSolvent

Tincture of Iodine

MaterialAmountFunction
Iodine70 gOxidant
Potassium iodide50 g
Distilled water50 mLSolvent
100% ethanol950 mLSolvent

Compounding Procedures

Tincture of iodine

  1. Mix the dry iodine and potassium iodide together.
  2. Add the water and mix well to dissolve.
  3. Add the ethanol and mix well.

Staining solution

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

Protocol

  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

  • Horneyold’s formula is a modification of Böhmer’s Alum hematoxylin.
  • This solution is regressive, and is recommended for osmium fixed tissue.
  • 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.
  • 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.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Bolles Lee, A.. Edited by Gatenby, J.B. and Beams, H.W., (1950)
    The Microtomist’s Vade-Mecum. 11 ed.,
    Churchill, London, UK.
  2. Lange, N.A., (1966)
    Lange’s Handbook of Chemistry, Revised 10th ed.,
    McGraw-Hill.