Routine Hematoxylin and Eosin (H&E): Regressive Method
Purpose
All tissues cut in a Histology lab receive a routine H&E stain. This stain is used for diagnosis, or to decide if any special stains are required to determine a final diagnosis for the patient so it is important the H&E is done properly and of optimal quality.
Principle
Hematoxylin is extracted from a tree and is not a dye. Once ripened with an oxidant (exposure to oxygen or to oxidizing agents) hematoxylin becomes hematein, a weakly acid dye. With the addition of a salt of a heavy metal (mordant), usually aluminum or iron, a mordant-dye “lake” is formed. The mordant-dye lake is now a basic acting stain with a positive charge (cationic) and will bind to negatively charged tissue elements (nuclei).
A regressive staining method involved overstaining the tissue with hematoxylin, and selectively removing from non-nuclear structures. This is accomplished with the use of a weak acid (1% HCl), in an alcoholic solution (70% ethanol). Differentiation of the hematoxylin is assessed by microscopic examination of the slide.
The counterstain is Eosin, an anionic dye (negatively charged) that binds to positively charged tissue elements (cytoplasm). Eosin staining will occur in varying intensities with different non-nuclear elements of the tissue, and excess is differentiated from the tissue using 95% alcohol.
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
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
Lithium Carbonate 1%
- Lithium Carbonate 1g
- Distilled Water 100 mL
Acid Alcohol 1% (used in a regressive method only) Prepare in fumehood!
- 70% Alcohol 99ml
- concentrated Hydrochloric Acid 1mL
Expiry: 6 months
Safety
- Hydrochloric acid is caustic and a respiratory irritant, use caution, flush with water. Remember: Acid into Water. Wear gloves, goggles and lab coat.
- Avoid contact with and inhalation of dyes.
Procedure-Regressive
Regressive method is over stained and decolorized by acid alcohol while progressive method is stained until the endpoint is reached.
- Dewax and rehydrate sections (take to water)
- Place sections in haematoxylin for 5 minutes
- Wash in running tap water (1 min.)
- Differentiate in 1% acid alcohol: 2 dips
- Wash immediately in running tap water (1 min)
- ‘Blue’ sections in lithium carbonate for 1 minute (do not agitate as sections could fall off slide in alkaline environment) or until sections are “blue”. You cannot “overblue” and are usually able to macroscopically see the change in color of the tissue once it is “blued”.
- Wash in running tap water. (1 min)
- Check under the microscope. Nuclei should be blue to blue-black. Any blue coloration of cytoplasm and connective tissue should be extremely faint.
- Place sections in alcoholic eosin (counterstain): 10-20 dips
- Dehydrate by using the following:
- 95% alcohol: 2 changes, 10 rapid dips each (will also pull out some eosin)
- Absolute alcohol: 3 changes, 10 dips each (should “stream” in the last alcohol)
- Formula 83: 3 changes 10 dips each (should “stream” in the last formula 83)
Results
- Nuclei: Blue to blue-black
- Cytoplasm: varying shades of pink
- Muscle fibres: Deep pink.
- Collagen: light pink
- Red blood cells: Orange/red
Control
Small intestine, appendix, or multi-tissue control