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Picro-Fuchsin

Herovici’s Stain for Young and Mature Collagen

By Dye Type, Picro-Fuchsin, Protocols, Stain Type, Trichrome Staining
Protocol

Herovici's Stain

for Young and Mature Collagen

7
steps
7
materials
print

Materials

Tissue Sample

Paraffin sections at 5µ of formol-acetic-ethanol (10:5:85) fixed tissue was recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with the acid resistant nuclear stain.
  3. Rinse well with tap water.
  4. Place into the staining solution for 2 minutes.
  5. Wash with 1% acetic acid for 2 minutes.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Young collagen and reticulin – blue
  • Mature collagen – red
  • Cytoplasm – yellow
  • Nuclei – black

Notes

  • Solution A is van Gieson’s solution.
  • Cook notes that the original included a final step with metanil yellow for cytoplasmic staining, which he omitted.

Safety Note

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

References

  1. Cook, H. C., (1974)
    Manual of Histological Demonstration Techniques
    Butterworths, London, UK.
    Citing:
    Herovici, c., (1963)
    Stain Technology, v. 38, p. 204
March 30, 2023
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Puchtler’s Picro-Sirius red for Collagen

By Dye Type, Picro-Fuchsin, Protocols, Stain Type, Trichrome Staining
Protocol

Puchtler's Picro-Sirius red

for Collagen

6
steps
4
materials
print

Materials

Picro-sirius red

MaterialAmount
Sirius red F3B0.5g
Saturated aqueous picric acid500mL

Acetic acid water

MaterialAmount
Acetic acid, glacial5mL
Distilled water11mL

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable.

Protocol

  1. Bring sections to water.
  2. Optionally, stain nuclei with an acid resistant nuclear stain.
  3. Wash well with water.
  4. Place into Picro-sirius red solution for 1 hour.
  5. Wash with two changes of acetic acid water.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

Light Microscopy

  • Nuclei – black if stained
  • Collagen – red
  • Cytoplasm – yellow

Polarising Microscopy

  • Large fibres – yellow or orange birefringence
  • Thin fibres – green birefringence

Notes

  • This method can be used as an alternative for van Gieson’s stain or, using polarising microscopy, as a sensitive method for collagen.

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.
March 30, 2023
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van Gieson’s Stain for Collagen

By Dye Type, Picro-Fuchsin, Protocols, Stain Type, Trichrome Staining
Protocol

van Gieson's Stain

for Collagen

7
steps
3
materials
print

Materials

Tissue Sample

Paraffin sections at 5µ are suitable. Many fixatives, including formalin, are satisfactory. Stains using acid dyes often benefit from picric acid or mercuric chloride fixation.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with the acid resistant nuclear stain.
  3. Rinse well with tap water.
  4. Place into the staining solution for 2-5 minutes.
  5. Optionally, rinse quickly with distilled water.
  6. Dehydrate with ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Collagen – red
  • Cytoplasm – yellow
  • Nuclei – black

Notes

  • This method is often used to counterstain other primary staining methods. In that case the nuclear stain may not be necessary and should be ommitted.

Safety Note

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

References

  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Drury, R.A.B. and Wallington, E.A., (1980)
    Carleton’s histological technique Ed. 5
    Oxford University Press, Oxford, UK.
  3. Culling C.F.A., (1974)
    Handbook of histopathological and histochemical techniques Ed. 3
    Butterworth, London, UK.
March 30, 2023
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