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Elastic Fibers

Basic Fuchsin–Picric Acid for Elastic Fibers

By Elastic Fibers, Protocols, Stain Target
Protocol

Basic Fuchsin–Picric Acid

for Elastic Fibers

6
steps
4
materials
print

Materials

Basic fuchsin

MaterialAmount
Basic fuchsin0.5g
Distilled water500mL

Picric acid

MaterialAmount
Saturated alcoholic picric acid12mL
Acetone1L

Tissue Sample

A 10% Formalin variant is suitable. Paraffin sections at 5µ are preferred.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in basic fuchsin for 1-2 minutes.
  3. Rinse with tap water, then acetone.
  4. Differentiate briefly with picro-acetone until tissue is just decolorised.
  5. Rinse with acetone.
  6. Clear with xylene and mount with a resinous medium

Expected Results

  • Elastic fibres  –  red
  • Other tissues  –  yellow

Notes

  • The Brown and Brenn picric acetone usually requires weighing semi-dry picric acid. The amount in the saturated ethanolic solution specified above is very close to that and it is a much safer means of compounding the solution.
  • If you use the Brown and Brenn variant of the Gram stain, the counterstain solutions from that may be used.
  • Be careful not to overdifferentiate. Apply picric acetone until most of the red is just removed from the background. This takes only a few seconds. It is easy to overdifferentiate.
  • This method is not meant as a primary means of staining elastic fibres. It can be done very quickly, about 5-10 minutes, and may be useful when time is limited.
  • Nuclei are not stained.

Safety Note

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

References

  1. Personal observation, Bryan D. Llewellyn.
  2. Brown, J H and Brenn, L, (1931),
    A method for the differential staining of Gram positive and Gram negative bacteria in tissue sections.,
    Bull. John Hopkins Hosp., v 48, page 69.
March 30, 2023
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Akita & Kaneko’s Hemalum for Elastic Fibers

By Dye Type, Elastic Fibers, Hematoxylin and Eosin Staining, Mordanted Hematoxylin, Protocols, Stain Target, Stain Type
Protocol

Akita & Kaneko's Hemalum

for Elastic Fibers

5
steps
6
materials
print

Materials

MaterialAmount
Hematoxylin100mg
Ethanol, 70%100mL
Sodium iodate20mg
Potassium alum300mg
Chloral hydrate5g
Citric acid100mg

Staining Solution Preparation

  1. Dissolve the hematoxylin into the ethanol.
  2. Add the potassium alum, then the other ingredients.
  3. Mix well for 15 to 20 minutes, then filter.
  4. At this point the solution is stable for 2 months at room temperature.
  5. Immediately prior to use, adjust to pH 8.0 with saturated aquueous lithium carbonate or 1N potassium hydroxide.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Helly and Bouin are also suitable. Other fixatives should be tested.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place into freshly pH adjusted staining solution for 30-60 minutes.
  3. Wash with running tap water for 10 minutes.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  blue
  • Collagen  –  pale violet

Notes

  • Note that this is an ethanolic variant of Mayer’s hemalum.
  • If sodium iodate is left out, the staining solution lasts 3-4 days, but the staining time is extended to 60 minutes.
  • The solution may also be made by diluting 30 mL Mayer’s hemalum with 70 mL absolute ethanol, mixing for 20 minutes, and filtering before adjusting the pH. Staining requires 60 minutes and the results are paler.

Safety Note

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

References

  1. Akita, M. and Kaneko, K., (1981)
    An improved hematoxylin method with application of Mayer’s hemalum for staining of elastic fibres.
    Stain Technology, v. 56, p. 327
  2. Kaneko, K. and Akita, M., (1978)
    On the staining of elastic fibres with alum hematoxylin.
    Stain Technology, v. 53, p. 43.
March 30, 2023
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Cook & Lamb Alkali Blue for Elastic Fibres

By Elastic Fibers, Protocols, Stain Target
Protocol

Cook & Lamb Alkali Blue

for Elastic Fibres

10
steps
3
materials
print

Materials

  • 0.4% alkali blue in 70% ethanol
  • 4% aqueous ferric ammonium sulphate
  • 0.05% potassium hydroxide in absolute ethanol

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. Place in alkali blue solution for 5 minutes.
  3. Wash with water.
  4. Place in iron alum solution for 3 minutes.
  5. Wash with water.
  6. Rinse with ethanol.
  7. Rinse briefly with alcoholic potassium hydroxide to clear the background.
  8. Counterstain if wished.
  9. Dehydrate with ethanols.
  10. Clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres – blue (blue-green after Van Gieson)
  • Erythrocytes – pale blue-green

Notes

  • Neutral red, safranin, hemalum and eosin are suggested as counterstains.
  • The alkali blue solution lasts for several weeks.
  • This is an example of afterchrome mordanting.

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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Gomori’s Aldehyde Fuchsin

By Aldehyde Fuchsin, Dye Type, Elastic Fibers, Intracytoplasmic Granules, Mast Cells, Protocols, Stain Target, Stain Type
Protocol

Gomori's Aldehyde Fuchsin

7
steps
4
materials
print

Materials

Solution A

MaterialAmount
Basic fuchsin1g
Paraldehyde, fresh1mL
Hydrochloric acid, conc.1mL
Ethanol, 70%200mL

Compounding Procedure

  1. Dissolve the dye in the ethanol.
  2. Add paraldehyde and hydrochloric acid.
  3. Ripen at room temperature for 48-72 hours.
  4. Refrigerate. The solution is stable for 2-3 months.

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. Wash with water.
  3. Rinse with 70% ethanol.
  4. Place in the staining solution for 10 minutes.
  5. Rinse well with 95% ethanol.
  6. Counterstain the nuclei and/or the cytoplasm if wished.
  7. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  purple
  • Mast cells  –  purple
  • Pituitary β cells  –  purple
  • Sulphated mucins  –  purple
  • Background  –  as the counterstain
  • Nuclei  –  as the nuclear stain

Notes

  • The basic fuchsin used for this solution should be one that is suitable for Schiff’s reagent, i.e., it should have a high pararosanilin content. Both methods involve forming a compound between an aldehyde and dye.
  • Light counterstaining with a progressive alum hematoxylin and eosin is also suitable.
  • Many other counterstains can be used, including methods such as Masson’s trichrome.
  • Gabe described a technique for the preparation and use of aldehyde fuchsin powder.

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.
March 30, 2023
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Gabe’s Aldehyde Fuchsin

By Aldehyde Fuchsin, Dye Type, Elastic Fibers, Protocols, Stain Target, Stain Type
Protocol

Gabe's Aldehyde Fuchsin

6
steps
9
materials
print

Materials

Stock powder

MaterialAmount
Pararosanilin5g
Paraldehyde5mL
Hydrochloric acid10mL
Distilled water1L

Dye Preparation Procedures

  1. Add the dye to the water and boil.
  2. Cool to room temperature.
  3. Add paraldehyde and hydrochloric acid.
  4. Ripen at RT two days. Filter. Wash precipitate with 50mL distilled water. Dry the ppt and paper at 60°C.
  5. Store in a labelled container. Stable for about ten years.

Working solution

MaterialAmount
Stock powder0.25g
Ethanol, 70%200mL
Acetic acid, glacial2mL

Acid ethanol

MaterialAmount
Hydrochloric acid, conc.1mL
Ethanol, 95%200mL

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. Place in the working solution for 5 minutes.
  3. Rinse well with running tap water.
  4. Rinse with acid ethanol for 20-30 seconds to remove excess dye.
  5. Counterstain if wished.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Elastic fibres  –  purple
  • Mast cells  –  purple
  • Pituitary βcells  –  purple
  • Sulphated mucins  –  purple
  • Background  –  as the counterstain
  • Nuclei  –  as the nuclear stain

Notes

  • The basic fuchsin used for this solution should be one that is suitable for Schiff’s reagent, i.e., it should have a high pararosanilin content. Both methods involve forming a compound between an aldehyde and dye.
  • Light counterstaining with a progressive alum hematoxylin and eosin is also suitable.
  • Many other counterstains can be used, including methods such as Masson’s trichrome.

Safety Note

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

References

  1. Kiernan. J.A., (1999)
    Histological and histochemical methods: Theory and practice, Ed. 3
    Butterworth Heinemann, Oxford, UK.
    Citing:
    Gabe, M., (1976)
    Histological techniques.
    Masson, Paris.
March 30, 2023
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