Rapid Hematoxylin & Eosin (Progressive Method)

purpose

A rapid H&E is often employed when the stained slide is needed more urgently, such as with intra-operative frozen sections.  The rapid method is validated to produce an acceptable quality slide in a minimum amount of time.

Note that other progressive H&E methods have been developed for routine use and the reagent times will vary.

principle

The principle of the progressive H&E is the same as the regressive method.  The difference is that in progressive methods there is no overstaining of nuclear material – the process is stopped once proper staining level has been achieved.  Therefore, there is no differentiation with acid-alcohol before the neutralization (bluing) step.

solutions

Harris’ haematoxylin

  • Haematoxylin – 5 g
  • 100% alcohol – 50 mL
  • Potassium alum – 100 g (mordant)
  • Distilled water – 1 L
  • Mercuric oxide – 2.5 g (alternatively, sodium iodate may be used as an oxidizer)
  • Glacial acetic acid – 40 mL

Dissolve the potassium alum in the water by warming and stirring. Dissolve the haematoxylin in the alcohol and add to the potassium alum. Bring rapidly to a boil and remove from heat. Add the mercuric oxide. Cool, add the acetic acid and filter. Ready for use immediately.

Note: We use purchased for student labs

 

Lithium Carbonate 1%

  • Lithium Carbonate 1g
  • Distilled Water 100 mL

 

Eosin Y (yellowish) alcoholic

  • Eosin Yellowish 1.0g
  • Distilled Water 100ml
  • Use 200 mls of the above 1% solution and mix with the following:
    • o 95% ethyl alcohol 600 mL
    • o Glacial acetic acid 4 mL

Note: We use purchased for student labs

Procedure – Progressive [1]
  1. For frozen sections: immediately fix for 1 min in 10% NBF, rinse well in distilled water.  For paraffin sections: dewax and hydrate section as per lab procedure.
  2.  Transfer slides to Harris hematoxylin, stain for 30 seconds
  3. Wash well in distilled water (10 dips)
  4. Transfer to lithium carbonate/bluing reagent  (10 dips)
  5. Wash well in distilled water (10 dips)
  6. Counterstain with 1% eosin for 20 seconds
  7. Begin dehydration with 2 changes of 95% alcohol (10 rapid dips each).  This will also differentiate the counterstaining.
  8. Complete dehydration with 3 changes of 100% alcohol (10 dips each)
  9. Clear slides in 3 changes of Formula 83 (10 dips each)
  10. Mount slides using Permount

Results

  • Nuclei: Blue
  • Erythrocytes/eosinophilic granules: Bright pink to red
  • Cytoplasm/other tissue elements: Various shades of pink

control

Bowel, appendix, or multi-tissue control


  1. https://www.newcomersupply.com/documents/staining/procedures/Hematoxylin%20&%20Eosin%20Stain%20Frozen%20Sections.nonkit.pdf and https://www.youtube.com/watch?v=UnCLhowHucU

License

Histotechnology Lab Manual Copyright © 2022 by NSCC. All Rights Reserved.

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