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
- 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.
- Transfer slides to Harris hematoxylin, stain for 30 seconds
- Wash well in distilled water (10 dips)
- Transfer to lithium carbonate/bluing reagent (10 dips)
- Wash well in distilled water (10 dips)
- Counterstain with 1% eosin for 20 seconds
- Begin dehydration with 2 changes of 95% alcohol (10 rapid dips each). This will also differentiate the counterstaining.
- Complete dehydration with 3 changes of 100% alcohol (10 dips each)
- Clear slides in 3 changes of Formula 83 (10 dips each)
- 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
- https://www.newcomersupply.com/documents/staining/procedures/Hematoxylin%20&%20Eosin%20Stain%20Frozen%20Sections.nonkit.pdf and https://www.youtube.com/watch?v=UnCLhowHucU ↵