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Eosinophils

Eosin for Eosinophils

By Eosinophils, Intracytoplasmic Granules, Protocols, Stain Target
Protocol

Eosin

for Eosinophils

6
steps
2
materials
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Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei lightly with alum hematoxylin.
  3. Differentiate if necessary and blue.
  4. Place in eosin solution for 5 minutes.
  5. Wash well with tap water until sections are almost unstained.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – blue
  • Eosinophils – bright pink
  • Background – pale pink to colorless

Notes

  • The tap water should be hard and very slightly alkaline.
  • Areas which have acidic or soft tap water should use Scott’s tap water substitute. If this is too alkaline, it may be diluted.
  • The water wash is to remove eosin from connective tissues so that the eosinophil granules are the most prominent feature. A light pink background helps with orientation.
  • Adjust staining and washing times to obtain suitable results.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

March 30, 2023
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Romanowsky – Giemsa General Oversight Stain

By Eosinophils, Intracytoplasmic Granules, Protocols, Stain Target
Protocol

Romanowsky – Giemsa

General Oversight Stain

9
steps
3
materials
print

Materials

  • Stock Giemsa or other Romanowsky stain e.g. Lieshman, Wright etc.
  • 0.1% acetic acid in distilled water.

Working Solution

MaterialAmount
Stock Romanowsky stain1mL
Distilled water or pH 6.8 buffer9mL

Make the diluted solution just before using. Discard after a few hours.

Tissue Sample

Most fixatives permit staining but the results may vary. 3 µ paraffin sections of neutral buffered formalin fixed tissue are usually suitable.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Optionally, treat with pH 6.8 phosphate buffer for 30 minutes.
  3. Place into the staining solution for 1 hour.
  4. Rinse well with water.
  5. Differentiate with acetic acid until nuclear morphology is clear. Control microscopically.
  6. Rinse well with distilled water.
  7. Blot dry with filter paper, then flood with xylene.
  8. Repeat step 6 until section is transparent (usually 4-5 times).
  9. Mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  pink

Notes

  • Giemsa is usually diluted 1 in 10. Lieshman, Wright and others are often diluted 1 in 3.
  • Pretreatment with a pH 6.8 phosphate buffer is sometimes recommended immediately before placing in diluted stain. In that case, buffer with the same pH should be used to dilute the stock Romanowsky stain.
  • Differentiation may also be carried out in the same buffer as used to dilute the stock stain, but may take some time.
  • Drury and Wallington use pH 5.0 buffer mixed equal parts with methanol, and specify green Euparal as the mounting medium.
  • Sections may be rapidly dehydrated with ethanol instead of blotting, but this removes some blue staining and will likely destroy any metachromasia.
  • Sections stained with Romanowsky stains do not usually show the same range of colors that are shown by the same stain on smears, and the choice of stock Romanowsky stain does not necessarily influence the final appearance.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Culling, C.F.A., Alison, R.T. and Barr, W.T. (1985)
    Cellular Pathology Technique, 4th ed.
    Butterworths, London, UK.
  2. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
March 30, 2023
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Lendrum’s Chromotrope 2R for Eosinophils

By Eosinophils, Intracytoplasmic Granules, Protocols, Stain Target
Protocol

Lendrum's Chromotrope 2R

for Eosinophils

6
steps
4
materials
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Materials

Preparation

  1. Place the phenol in an Erlenmeyer flask and melt it under hot water through the glass.
  2. Add the chromotrope 2R and mix well into a sludge.
  3. Add the water and mix well.
  4. Filter before use.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with Mayer’s hemalum and blue.
  3. Place in the staining solution for 30 minutes.
  4. Wash well with tap water.
  5. Dehydrate with ethanol.
  6. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Eosinophil granules  –  bright red
  • Erythrocytes  –  may be lightly stained
  • Paneth cell granules  –  may be brownish
  • Enterocromaffin granules  –  may be brownish

Notes

  • The basis of this method is difficult to rationalise. Phenol is acidic and thus lowers the pH. This is often used as the basis for explaining the method, but usually an acid dye stains all basic components of the tissue (muscle, cytoplasm, collagen) intensely when applied at an acid pH. With this method eosinophils are intensely stained, but other components that would usually stain with an acidified acid dye are not. Phenol can have an intensifying effect, as is clear from its inclusion in carbol fuchsin, when it intensifies staining with basic fuchsin, but the mechanism has not been satisfactorily explained.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Histological demonstration techniques, (1974))
    Cook, H C.
    Butterworths, London, England.
March 30, 2023
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Llewellyn’s Sirius Red for Amyloid

By Amyloid, Direct Dye Staining, Eosinophils, Intracytoplasmic Granules, Paneth Cells, Protocols, Stain Target, Stain Type
Protocol

Llewellyn's Sirius Red

for Amyloid

8
steps
3
materials
print

Materials

MaterialAmount
Sirius red F3B0.5g
Distilled water50mL
Ethanol, absolute50mL

Staining Solution Preparation

  1. Dissolve the dye into the water, add ethanol and mix well.
  2. Add 1 mL of 1% sodium hydroxide. Then, while strong backlighting and swirling, add drops of 20% sodium chloride until a fine haze is detected. Usually about 2 mL is adequate. Adding more than 4 mL causes excessive precipitation. The solution is reasonably stable for several months, but slowly deteriorates. Extend the staining time to compensate. When it requires more than 2 hours to adequately stain, prepare a new solution.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with a progressive alum hematoxylin for a few minutes.
  3. Rinse with tap water.
  4. Rinse with ethanol.
  5. Place into alkaline sirius red for 1 – 2 hours.
  6. Rinse well with tap water.
  7. Dehydrate with absolute ethanol.
  8. Clear with xylene and mount with a resinous medium.

Expected Results

  • Amyloid  –  red
  • Eosinophil and Paneth cell granules  –  red
  • Nuclei  –  blue
  • Background  –  colorless
   

Notes

  • Amyloid displays deep green birefringence when viewed with crossed polarisers, one above and one below the section.
  • Eosinophils and Paneth cell granules are also demonstrated. If used for this purpose the sodium chloride may be ommitted.
  • This method uses sirius red F3B. The dye Sirius red 4B is not suitable.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.

References

  1. Llewellyn, B.D., (1970)
    An improved sirius red method for amyloid.
    Journal of Medical Laboratory Technology, v 23, 308
March 30, 2023
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