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Hematoxylin and Eosin Staining

Staining of Sections for Histology

Place slides in slide holder and have all of solutions pre-made and in dipping trays prior to starting staining procedure. First two xylene washes are very important for complete de-paraffinization of the sections.

  • Xylene: 3-5 minutes, 2X
  • 100% Ethanol: 3-5 minutes, 2X
  • 95% Ethanol: 3-5 minutes
  • 85% Ethanol: 3-5 minutes
  • 70% Ethanol: 3-5 minutes
  • Eosin*: 15-30 seconds
  • 50% Ethanol: 3-5 minutes
  • 30% Ethanol: 3-5 minutes
  • H2O: 3-5 minutes
  • Hematoxylin: 10 seconds to 2 minutes, depending on tissue
  • H2O: 3-5 minutes
  • 30% Ethanol: 3-5 minutes
  • 50% Ethanol: 3-5 minutes
  • 70% Ethanol: 3-5 minutes
  • 85% Ethanol: 3-5 minutes
  • 95% Ethanol: 3-5 minutes
  • 100% Ethanol: 3-5 minutes, 2X
  • Xylene: 3-5 minutes, 2X

*Eosin

  • 2.5 ml Acetic Acid
  • 0.5 g Eosin
  • 497.5 ml 70% Ethanol

Fixing Slides

  • Blot away excess xylene, add 2-3 drops Permount and place coverslip on slide. Push out air bubbles

    This should dry in ~10 min, but wait until the next day to place on microscope.
  • You can also clean away extra Permount using xylene

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