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Yellowsolve Staining

Lendrum’s Phloxine Tartrazine for Viral Inclusion Bodies

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

Lendrum's Phloxine Tartrazine

for Viral Inclusion Bodies

11
steps
6
materials
print

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.
March 30, 2023
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Modified Yellowsolve for General Oversight

By Protocols, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Modified Yellowsolve

for General Oversight

10
steps
8
materials
print

Materials

  • Hemalum
  • Trichlorethylene
  • Solution A
    MaterialAmount
    Phloxine B0.5g
    Calcium chloride0.5g
    Distilled water100mL
  • Solution B
    MaterialAmount
    Luxine pure yellowtosaturation
    2-Ethoxyethanol50mL
    Ethyl phosphate50mL

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Lendrum says, “If … the time of differentiation is kept short the result is comparable to the best examples of the old Masson’s erythrosin-saffron technique.” Masson’s erythrosin-saffron is the same technique as the HPS (hematoxylin-phloxine-saffron) using phloxine instead of erythrosin.

Protocol

  1. Dewax sections with xylene.
  2. Place in trichlorethylene for 24 to 48 hours.
  3. Bring sections to water.
  4. Stain nuclei with hemalum, differentiate and blue.
  5. Wash well with water.
  6. Place in solution A for 30 minutes.
  7. Rinse with 2-ethoxyethanol.
  8. Differentiate with solution B, controlling microscopically, until collagen is yellow.
  9. Rinse with 2-ethoxyethanol.
  10. Clear in xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Muscle  –  red
  • Background  –  yellow

Notes

  • Stop differentiation when collagen is yellow and muscle is still red.
  • 2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH3CH2OCH2CH2OH.
  • Trichlorethylene has the formula ClHC=CCl2 or C2HCl3
  • Trichlorethylene should be used in a fume hood.

Safety Note

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

References

  1. Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
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Slidders’ Fuchsin-Miller for Fibrin

By Fibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Slidders’ Fuchsin-Miller

for Fibrin

16
steps
7
materials
print

Materials

  • An acid resistant nuclear stain, such as Weigert’s iron hematoxylin, or the celestine blue-hemalum sequence.
  • Fuchsin
    MaterialAmount
    Acid fuchsin1g
    Acetic acid, glacial2.5mL
    Distilled water100mL
  • Miller
    MaterialAmount
    Milling yellow 3G2.5g
    2-ethoxy-ethanol100mL
  • Phosphotungstic acid
    MaterialAmount
    Phosphotungstic acid1g
    Distilled water100mL

Tissue Sample

3 mm slices of tissue should be fixed in formol sublimate or B5 overnight. Paraffin process overnight. Overnight formalin fixation and paraffin processing can produce acceptable results if sections are refixed for an hour in Bouin’s picro-acetic-formalin mixture at 56°C. Avoid rapid fixation with formalin and short processing, as this produces tissues that stain poorly even with the secondary fixation specified. Optimal results are obtained with extended mercuric chloride fixation, thorough processing, degreasing and secondary fixation of sections. Sections should be 3-5 µ thick.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. If mercury fixed, remove with the iodine-thiosulphate sequence.
  3. If not mercury fixed, place into Bouin’s fluid at 56°C for 1 hour.
  4. Rinse well with distilled water.
  5. Stain nuclei with an acid resistant nuclear stain.
  6. Rinse with 95% ethanol.
  7. Place in fuchsin for 10 minutes.
  8. Rinse with distilled water.
  9. Differentiate with phosphotungstic acid for 5 minutes.
  10. Rinse well with distilled water.
  11. Blot.
  12. Rinse well with cellosolve.
  13. Place into milling yellow until fibrin is red and tissues are yellow.
    This step takes from ½ – 4 hours.
  14. Rinse briefly with 1% aqueous acetic acid.
  15. Rinse with cellosolve.
  16. Clear with xylene and mount with a resinous medium.

Expected Results

  • Fibrin  –  red
  • Other tissue  –  yellow
  • Nuclei  –  black

Notes

  • It is very important that the milling yellow solution be completely anhydrous. Even small amounts of water or other solvents can cause problems with displacement and incomplete removal of red dye. For that reason, step 12 should be thorough and the milling yellow should be used in a Coplin jar with a lid sealed with tape.
  • A well stained section would show red fibrin only, muscle and erythrocytes being yellow. With poorly fixed material, both erythrocytes and muscle fibres may resist displacement of the red dye and they may have some residual red colouring. Sometimes this may be overcome by prestaining either with saturated picric acid in absolute ethanol or the MSB martius yellow solution for two minutes immediately before step 7, but a better resolution is correct fixation and processing.
  • Some intracellular materials may stain red, such as Paneth cell granules.

Safety Note

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

References

  1. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  2. Bancroft, J.D. and Stevens A. (1982)
    Theory and practice of histological techniques Ed. 2
    Churchill Livingstone, Edinburgh & London, UK.
March 30, 2023
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Yellowsolve I for Fibrin

By Fibrin, Protocols, Stain Target, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Yellowsolve I

for Fibrin

13
steps
9
materials
print

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Protocol

  1. Dewax sections with xylene.
  2. Place in trichlorethylene for 24 to 48 hours.
  3. Bring sections to water.
  4. Stain elastic fibres if desired.
  5. Stain nuclei with the celestine blue-hemalum sequence.
  6. Wash well with water.
  7. Place in solution A for 30 minutes.
  8. Rinse with 2-ethoxyethanol then with tetrachlorethylene.
  9. Place into tetrachlorethylene overnight.
  10. Rinse with 2-ethoxyethanol.
  11. Differentiate with solution B, controlling microscopically.
  12. Rinse with 2-ethoxyethanol.
  13. Clear in xylene and mount with a synthetic resinous medium.

Expected Results

  • Fibrin  –  red
  • Background  –  yellow
  • Nuclei  –  black
  • Acidophil cytoplasmic inclusions  –  red

Notes

  • 2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH3CH2OCH2CH2OH.
  • Tetrachlorethylene has the formula Cl2C=CCl2 or C2Cl4
  • Trichlorethylene has the formula ClHC=CCl2 or C2HCl3
  • Both trichlorethylene and tetrachlorethylene should be used in a fume hood.

Safety Note

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

References

  1. Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
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HPS Variant General Oversight Stain

By Protocols, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

HPS Variant

General Oversight Stain

10
steps
5
materials
print

This is also known as the Hematoxylin Phloxine Saffron stain.

Materials

  • Regressive hemalum
  • Solution A
    MaterialAmount
    Phloxine B2g
    Distilled water100mL

    Dissolve the phloxine B and filter. Preserve with a crystal of thymol.

  • Solution B
    MaterialAmount
    Saffron30g
    Absolute ethanol1L

    Add the saffron to 500ml absolute ethanol and extract at 60°C for 48 hours. Decant, and store in a dark bottle. Repeat with 500 mL fresh absolute ethanol and combine with the first extraction.

Tissue Sample

No fixative was specified, but most methods using acid dyes benefit from picric acid or mercuric chloride fixation. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Treat with hemalum for 5 minutes, differentiate and blue.
  3. Wash with running tap water for 5 minutes.
  4. Place in solution A for 5 minutes.
  5. Wash with running tap water for 5 minutes.
  6. Differentiate phloxine with 95% ethanol.
  7. Thoroughly dehydrate with absolute ethanol.
  8. Place into solution B for 5-10 minutes until a suitable red-yellow balance is achieved.
  9. Dehydrate with 4 changes of absolute ethanol.
  10. Clear with 4 changes of xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei – blue
  • Cytoplasm – red shades
  • Collagen – yellow

Notes

  • Harris’ hemalum was named, but the formula given was a variant of Bencosme’s hemalum.
  • Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring. Usually, whole stigmata are more effective than ground saffron.
  • Store and use the saffron solution at room temperature. It has a limited life of about a month and is best when freshly made. It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied. If contaminated with moisture the solution must be discarded.

Safety Note

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

References

  1. Histonet posting
March 30, 2023
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Yellowsolve II for Cellular Inclusions

By Protocols, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Yellowsolve II

for Cellular Inclusions

10
steps
7
materials
print

This method is also known as the Rhoda-Coomassie.

Materials

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are likely suitable. It should be noted, however, that the authors of this method favoured extended fixation in formal sublimate of up to 10 days for fibrin. This fixative is now deprecated due to its mercuric chloride content. With other staining methods, pretreatment of sections with Bouin’s fluid for an hour at 60°C can compensate for the lack of mercury fixation to a large degree.

Protocol

  1. Dewax sections with xylene.
  2. Place in trichlorethylene for 24 to 48 hours.
  3. Bring sections to water.
  4. Stain nuclei with the celestine blue-hemalum sequence.
  5. Wash well with water.
  6. Place in solution A for 5 minutes.
  7. Rinse with 2-ethoxyethanol.
  8. Differentiate with solution B, controlling microscopically.
  9. Rinse with 2-ethoxyethanol.
  10. Clear in xylene and mount with a synthetic resinous medium.

Expected Results

  • Cell inclusions  –  red
  • Background  –  yellow
  • Nuclei  –  black

Notes

  • Inclusions stained may include mast cells, plasma cells and Paneth cells as well as fibrin.
  • 2-Ethoxyethanol is also known as cellosolve or ethylene glycol monoethyl ether, and has the formula CH3CH2OCH2CH2OH.
  • Trichlorethylene has the formula ClHC=CCl2 or C2HCl3
  • Trichlorethylene should be used in a fume hood.

Safety Note

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

References

  1. Lendrum A C, Fraser D S, Slidders W and Henderson R. (1962)
    Studies on the character and staining of fibrin.
    Journal of clinical pathology, v. 15, p. 401.
March 30, 2023
Love0

HPS, AFIP Modification General Oversight stain

By Protocols, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

HPS, AFIP Modification

General Oversight stain

11
steps
6
materials
print

Also known as the Hematoxylin Phloxine Saffron stain, AFIP modification.

Materials

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable, as are many other fixatives.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place in picric acid solution for 5 minutes.
  3. Wash with water to remove yellow.
  4. Stain nuclei with hemalum, differentiate and blue.
  5. Wash well with water.
  6. Place in solution A for 2 minutes.
  7. Wash with tap water for 5 minutes.
  8. Thoroughly dehydrate with absolute ethanol.
  9. Place into solution B for 5 minutes.
  10. Rinse well with absolute ethanol.
  11. Clear in xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Muscle & cytoplasm  –  red
  • Collagen  –  yellow

Notes

  • The saffron should be extracted by mixing with anhydrous ethanol in a tightly capped container, then placing in a 56°C oven for a few days. Store and use at room temperature. It has a limited life and is best when freshly made.
  • Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring.
  • It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied.
  • Usually, whole stigmata are more effective than ground saffron.
  • Erythrosine B may be used instead of phloxine B.

Safety Note

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

References

  1. Histological Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. (1968)
    Luna, Lee G.
    McGraw-Hill, NY, USA
March 30, 2023
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Bencosme’s HPS General Oversight stain

By Protocols, Stain Type, Trichrome Staining, Yellowsolve Staining
Protocol

Bencosme's HPS

General Oversight stain

11
steps
5
materials
print

Also known as the Hematoxylin Phloxine Saffron stain.

Materials

  • Regressive hemalum
  • Solution A
    MaterialAmount
    Phloxine B2g
    Distilled water100mL

    Dissolve the phloxine B and filter. Preserve with 2-5 drops of strong formalin.

  • Solution B
    MaterialAmount
    Saffron6g
    Absolute ethanol200mL

    Mix the saffron stigmata with the ethanol and seal tightly. Place in an oven at 56°C for 1-2 weeks.

Tissue Sample

Brasil’s fixative was specified. 5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable, but results may be improved by refixing in Bouin’s fixative or saturated aqueous picric acid at 56°C for one hour prior to staining, then washing the sections in tap water to remove all yellow discoloration.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Treat with Bouin’s fluid if desired.
  3. Stain nuclei with hemalum, differentiate and blue.
  4. Wash well with water.
  5. Place in solution A for 2-20 minutes.
  6. Wash with tap water until red stain stops being removed, 1-5 minutes.
  7. Differentiate phloxine with 80% ethanol up to 1½ minutes.
  8. Thoroughly dehydrate with absolute ethanol.
  9. Place into solution B for 2-20 minutes until a suitable red-yellow balance is achieved.
  10. Rinse well with absolute ethanol.
  11. Clear with xylene and mount with a synthetic resinous medium.

Expected Results

  • Nuclei  –  blue
  • Cytoplasm  –  red shades
  • Muscle  –  pink
  • Elastic fibres  –  bright red
  • Collagen  –  orange-yellow

Notes

  • Any hemalum may be used, but Bencosme specified a particular formula.
  • Saffron is expensive. It may be available in East Indian grocery stores or health food stores as its most common use today is as a spice and food coloring. Usually, whole stigmata are more effective than ground saffron.
  • If the saffron solution is too strong it may be diluted with absolute ethanol. Store and use at room temperature. It has a limited life of about a month and is best when freshly made. It is important that there be no water in the saffron solution and that sections be thoroughly dehydrated before it is applied. If contaminated with moisture the solution must be discarded.

Safety Note

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

References

  1. Medical Laboratory Technology, 2nd ed. (1969)
    Lynch M.J., Raphael S.S., Mellor L.D., Spare P.D. and Inwood M.J.H.,
    W. B. Saunders Co., Toronto, On., Canada
March 30, 2023
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