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Humberstone’s Stain for Elastic Fibres

Humberstone's Stain

for Elastic Fibres



  • Weigert’s iron hematoxylin or equivalent
  • Van Gieson’s picro-fuchsin
  • Potassium permanganate, 0.25% aqu., for the Mallory bleach
  • Humberstone’s solution
    Victoria blue 4R1g
    Ethyl violet1g
    Ferric chloride, 30% aqu.25mL
    Ethanol, 95%150mL
    Hydrochloric acid, conc.4mL
    Distilled water200mL
    • Preparation

      1. In an oversized flask, add the dyes to the water, and boil to dissolve.
      2. Add the resorcin, dextrin and ferric chloride. Boil for 3 minutes. Cool and filter.
      3. Place the precipitate and filter paper back into the original flask. Add 100 ml of 95% ethanol and ently for 15 minutes.
      4. Cool and filter.
      5. Make up the volume to 350 mL with 95% ethanol.
      6. Add the phenol and hydrochloric acid.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.


  1. Bring sections to water via xylene and ethanol.
  2. Do a Mallory bleach with 0.25% potassium permanganate for ten minutes.
  3. Rinse with 95% ethanol.
  4. Place into Humberstone’s solution overnight.
  5. Wash with 95% ethanol to remove excess solution.
  6. Wash in water.
  7. Counterstain with iron hematoxylin and van Gieson.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue-black
  • Nuclei  –  black
  • Cytoplasm  –  yellow
  • Collagen  –  red


  • The authors state that buffered formalin is an unsuitable fixative. I have not found this so and have used this method regularly with such tissue. It may be that the modern practice of minimalist fixation limits the effect.
  • As the solution ages, the demonstration of elastic fibres improves in both quality and speed. When the solution is a few years old elastic is demonstrated within about 4 hours, although overnight is generally
  • The solution is stable for more than ten years. Due to the improvement in staining as the solution ages, it is suggested that a litre, or more, be made at a time.
  • The authors suggest as suitable counterstains, iron hematoxylin and van Gieson, Masson’s trichrome or MSB (with green collagen).

Safety Note

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


  1. Humberstone, G. C. W., and Humberstone, F. D., (1969)
    An Elastic Tissue Stain
    Journal of Medical Laboratory Technology, V. 26, No 2, pp. 99.