H and E staining

Hematoxylin and Eosin staining of Tissue Section


Hematoxylin and Eosin staining of tissue section is main staining method of histopathology staining. It helps to identify human tissue structure.

The method of hematoxylin and eosin stains are described in short.


  • Put the slide on hot plate/ flaming for few seconds ( to soften paraffin )
  • Then immediately dip slide in XYLENE for 5 min.(Minimum) to dissolve paraffin. After taking slide out, put some fresh xylene (Few drops) on the section to washout remaining paraffin.
  • Wash slide with 100% alcohol in slanting way over both side of slide to remove xylene
  • Then wash the slide with descending grade of alcohol ie, 90%,-70%- 50%-(2 min each), to hydrate the slide
  • Ultimately put the slide in water for 5 min
  • Bring up the slide from water and put in Koplins jar containing Harris’ Hematoxylene for 5 min. ( in modified method put hematoxylene on tissue section for 5 min)
  • Keep slide in rectangular koplin jar in running water for 5 min.
  • Dip 6the slide for 2/3 times in 1% acid alcohol (1% HCL),for differentiation (To remove excess stain in cytoplasm. colour  appear pink.
  • Keep the slide in running water again till it become blue colour (Colour of the nucleus)
  • Put 1% aquous eosin over slide for 2 min.
  • Dip in water for 2 min, (in running water) or more if staining is more.  ( 30 SEC. IN CASE OF SPIRITE EOSIN SOLUTION)
  • Then dehydrate slide by putting ascending grade of alcohol ie, 50%-70%-90%-Absolute alcohol  for 2 min each.
  • Dry in air.
  • Dip the slide in clean xylene for 1 min
  • Mount in Canada Balsam/DPX
For practical purpose, blotting is done by tissue paper in place of  sl. 12


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