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Kefalas’s Iron Hematoxylin

Kefalas's Iron Hematoxylin

16
steps
4
materials

Materials

Solution A

MaterialAmountFunction
Hematoxylin1 gDye
Ferric chloride1 gMordant
Acetone100 mLSolvent
Hydrochloric acid0.05 mLAcidifier

Compounding Procedure

  1. Dissolve the ferric chloride and hematoxylin in the acetone.
  2. Add the hydrochloric acid.

Protocol

Standard Method

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 30 minutes.
  3. Wash well in running tap water to blue.
  4. Rinse with distilled water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Alternative Method

Since this solution is almost anhydrous it can be used in a staining procedure which completely avoids water or ethanol.

  1. Remove wax with xylene.
  2. Remove xylene with a few changes of acetone.
  3. Place into staining solution for 30 minutes.
  4. Wash with acetone.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • 0.05 mL hydrochloric acid would be about 1-2 drops.
  • The simplicity of the formula indicates it is likely not stable for long.
  • The presence of hydrochloric acid may indicate progressive staining.
  • It is likely suitable as an acid resistant nuclear stain.
  • Although the staining time should be established by trial and error, 30 minutes would likely suffice as a starting point.

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
    Citing:
    Kefalas, (1926),
    Journal of the Royal Microscopical Society
    v. 46, p. 277.