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Debiden’s Alum Hematoxylin Variants

Debiden's Alum Hematoxylin Variants

8
steps
6
materials

Materials

MaterialVariantFunction
19871991
Hematoxylin5 g5 gDye
Potassium alum100 g100 gMordant
Distilled water1 L1 LSolvent
Glacial acetic acid20 mL20 mLAcidifier
Mercuric oxide, red2.5 gOxidant
Javex (5.25% sodium hypochlorite)2 mLOxidant

Compounding procedures

  1. Dissolve the alum in the water in a large flask.
  2. Add the hematoxylin. Mix well.
  3. Place in a 60-65°C oven overnight.
  4. Remove from the oven, and add the appropriate oxidizing agent.
  5. Stir until cold. Filter.

Preparation notes

1980

  • Acetic acid may be added as soon as it is cooled following preparation.
  • It is usable immediately.
  • It is preferable to prepare small volumes monthly.

1991

  • This improves in staining ability if allowed to ripen for a week or two.
  • If acetic acid is used, it should be added just before use.

Protocol

  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

  • These two solutions are modifications of Harris’ hematoxylin and are regressive.
  • Paraffin sections should be stained for 5 minutes, and cytology smears for 45 seconds.
  • As with many strong regressive formulae, mucin may be stained blue.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • 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. Debiden, D., (1987)
    Improved preparation of Harris’ hematoxylin
    Histologic, v.18, No.8
  2. Debiden, D., (1991)
    A new oxidant for Harris’ hematoxylin
    Histologic, v.21, No.2