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Llewellyn’s Mordant Blue 3 for Nuclei

Llewellyn's Mordant Blue 3

for Nuclei

9
steps
8
materials

This is a substitute for alum hematoxylin in the H&E stain.

Materials

  • An aqueous or alcoholic eosin Y solution
  • Solution A
    MaterialVariant
    19741978a1978b
    Mordant blue 30.25g1g1g
    Iron alum, 10% aqu.40mL90mL5mL
    Sulphuric acid5mL
    Hydrochloric acid10mL
    Acetic acid50mL
    Distilled water955mL860mL985mL

    For all variants:

    1. Dilute the iron alum with the water.
    2. Add the mordant blue 3 and dissolve.
    3. Add the specified acid, mix and filter.

    The solutions are usable immediately and they have a shelf life of more than one year.

  • Solution B
    MaterialVariant
    Var AVar B
    Hydrochloric acid, conc1mL1mL
    Ethanol, 70%99mL
    Distilled water99mL
  • Solution C
    MaterialAmount
    Sodium acetate0.5g
    Distilled water100mL

Tissue Sample

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

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into solution A for 5 minutes.
  3. Rinse well with tap water.
  4. Differentiate with a variant of solution B:
    1. For the 1974 formula: var A for 1-3 seconds or var B for 30 seconds.
    2. For the 1978a formula: var A for about ten seconds.
    3. For the 1978b formula: do not differentiate. Proceed to step 6.
  5. Rinse well with tap water.
  6. Blue with solution C for about 30 seconds.
  7. Rinse well with tap water.
  8. Counterstain with an eosin solution.
  9. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  shades of pink

Human kidney stained progressively with mordant blue 3 and eosin Y at 40x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 40x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 100x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 100x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 400x magnification

Human kidney stained progressively with mordant blue 3 and eosin Y at 400x magnification

Notes

  • At times hematoxylin powder has become difficult, if not impossible, to buy. Due to the importance of this dye in making alum hematoxylin for H&E stains, several other dyes were investigated as substitutes. Mordant blue 3 was one of these, and Llewellyn published three variants of this substitute, both progressive and regressive.
  • The 1974 and 1978a formulae are regressive. The 1978b formula is progressive.
  • The sections will be blue after solution A. The blueing referred to in step 6 will deepen this, but it’s main purpose is to remove any pink staining due to unmordanted dye.
  • Iron alum is ferric ammonium sulphate:  FeNH4(SO4)2·12H2O. Use clean crystals.

Safety Note

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

References

  1. Llewellyn, B. D., 1974
    Mordant blue3: A readily available substitute for hematoxylin
    in the routine hematoxylin and eosin stain.
    Stain Technology, v. 49, pp. 347.
  2. Llewellyn, B. D., 1978
    Improved nuclear staining with mordant blue 3 as a hematoxylin substitute
    Stain Technology, v. 53, pp. 73.