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Paneth Cells

Lendrum’s Phloxine Tartrazine for Viral Inclusion Bodies

By Intracytoplasmic Granules, Paneth Cells, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining

Lendrum's Phloxine Tartrazine

for Viral Inclusion Bodies

11
steps
6
materials

This method is also used for the demonstration of Paneth cell granules, and may be used as a substitute for the HPS if the differentiation in the tartrazine solution is shortened to retain pink cytoplasm and muscle.

Materials

  • Mayer’s hemalum
  • Solution A
    MaterialAmount
    Phloxine B0.5g
    Calcium chloride0.5g
    Distilled water100mL
  • Solution B
    MaterialAmount
    Tartrazineto saturation
    2-Ethoxy ethanol (cellosolve)100mL

Tissue Sample

5µ paraffin sections of formal sublimate fixed tissue is preferred. Formalin fixed tissue is suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei to medium density with hemalum.
  3. Wash in tap water for 5 minutes. Blueing takes place in the phloxine solution.
  4. Place in solution A for 20 minutes.
  5. Rinse in tap water, blot almost dry. Some technologists rinse with cellosolve instead of blotting. The object is to remove all traces of water, as it interferes with the ability of tartrazine to extract phloxine and counterstain.
  6. Rinse with solution B to remove remaining water. Discard solution.
  7. Place in solution B until inclusions are red and all other tissue is yellow. The time varies considerably. Control microscopically.
  8. Rinse thoroughly but briefly with absolute ethanol.
    Do not rinse with water at this stage as it rapidly removes the tartrazine. Some technologists use cellosolve instead of ethanol.
  9. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Acidophil virus inclusion bodies  –  red
  • Paneth cell granules  –  red
  • Background  –  yellow

Notes

  • The 2-ethoxy ethanol must not be replaced by any other solvent.
  • The 2-ethoxy ethanol must be, and must remain, completely anhydrous.

Safety Note

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

References

  1. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.

Llewellyn’s Sirius Red for Amyloid

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

Llewellyn's Sirius Red

for Amyloid

8
steps
3
materials

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