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Graham’s Aluminum Hematoxylin

Graham's Aluminum Hematoxylin

6
steps
5
materials

Grahams Alum hematoxylin is a modification of Gill’s Alum hematoxylin, and was designed for routine staining of plant tissue. It should also be satisfactory for animal tissues. It has been formulated so that small quantities may be prepared fresh and sufficient for a single Coplin jar.

Materials

MaterialAmountFunction
Distilled water750 mLSolvent
Propylene glycol210 mLSolvent
Glacial acetic acid20 mLAcidifier
Aluminum sulphate17.6 gMordant
Sodium iodate0.2 gOxidant
MaterialAmountFunction
Solution BAmountFunction
Hematoxylin10 gDye
Propylene glycol100 mLSolvent

Compounding Procedures

Solution A

  1. Combine all the ingredients and filter when dissolved.

Solution B

  1. Shake vigorously several times during 48 hours until the hematoxylin dissolves.

Working solution

  1. Solution A – 49 mL
  2. Solution B – 1 mL
  3. Mix well.
  4. It is ready for use when an opaque dark purple.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • Staining is progressive, application time being determined by trial (try 2 minutes).
  • Propylene glycol replaces toxic ethylene glycol.
  • 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.
  • The aluminum sulphate should be Al2(SO4)3.18H2O. Adjust the amount if different.

Safety Note

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

References

  1. Graham, E. T., (1991)
    A quick mix aluminum hematoxylin stain
    Biotechnic and Histochemistry, pp. 279-281