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Protocols

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Kiernan’s Eriochrome Cyanin with two colours

By Dye Type, Hematoxylin Alternatives, Protocols, Stain Type
Protocol

Kiernan's Eriochrome Cyanin

with two colours

5
steps
4
materials
print

Materials

Solution A

MaterialAmount
Eriochrome cyanine R1.0g
Ferric chloride, 5.6%2.5mL
Sulphuric acid, conc.2.5mL
Distilled waterup to 500mL

Tissue Sample

5 µ 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 sections into solution A for 3 minutes.
  3. Rinse with distilled water three times for about 20 seconds each time.
  4. Dehydrate with ethanols.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  purple blue
  • Background  –  red

Notes

  • Solution A is stable for years.
  • Solution A must have a pH of 1.5 for the two colour effect. Check periodically, and adjust with 1M sulphuric or hydrochloric acids, or sodium hydroxide.
  • Kiernan’s also describes regressive nuclear staining and myelin staining variants.
  • Eriochrome cyanine R is also known as mordant blue 3 and solochrome cyanine R

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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Klatskin’s Modification of Masson’s Trichrome for Liver

By Protocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

Klatskin's Modification of Masson's Trichrome

for Liver

12
steps
8
materials
print

Materials

  • Harris’ alum hematoxylin.
  • Solution A
    MaterialAmount
    Picric acidto saturation
    Ethanol, 95%100mL
  • Solution B
    MaterialAmount
    Xylidine ponceau1g
    Distilled water99mL
    Glacial acetic acid1mL
  • Solution C
    MaterialAmount
    Phosphomolybdic acid1g
    Distilled water100mL
  • Solution D
    MaterialAmount
    Aniline blueto saturation
    Glacial acetic acid2.5mL
    Distilled water97.5mL
  • Solution E
    MaterialAmount
    Acetic acid, glacial1mL
    Distilled water99mL

Tissue Sample

3-5 micron paraffin sections of formalin fixed liver.

Protocol

  1. Bring sections to water via xylene and ethanol
  2. Stain with Harris’ hemalum for 10 minutes.
  3. Rinse with 95% ethanol.
  4. Place in solution A for 10 minutes.
  5. Wash with water for 10 minutes.
  6. Place in solution B for 5 minutes.
  7. Place in solution C for 5 minutes.
  8. Place in solution D for 2 minutes.
  9. Replace in solution C for 5 minutes.
  10. Place in solution E for 5 minutes.
  11. Place in 70% ethanol for 2 minutes.
  12. Dehydrate with ethanol, clear and mount with a resinous medium.

Expected Results

  • Cytoplasm  –  red
  • Erythrocytes  –  red
  • Muscle  –  red
  • Collagen  –  blue
  • Nuclei  –   brown

Notes

  • Aniline blue, is a mixture of two dyes, methyl blue, and water blue. Either may usually be substituted satisfactorily.
  • Although recommended for liver sections, this modification may also be used as a general replaceement for Masson’s method.

Safety Note

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

References

  1. Petersen, K.F., West, A.B., Reuben, A., Rothman, D. and Shulman, G.I. (1996)
    Noninvasive Assessment of Hepatic Triglyceride Content in Humans With 13C Nuclear Magnetic Resonance Spectroscopy.
    Hepatology, V. 24, p 114-117
March 30, 2023
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Koneff’s Trichrome for Pituitary Cells

By Protocols, Stain Type, Trichrome Staining, Trichrome, Multi-Step
Protocol

Koneff's Trichrome

for Pituitary Cells

17
steps
11
materials
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Materials

Solution A

MaterialAmount
Aniline0.1mL
Ethanol, 90%100mL

Solution B

MaterialAmount
Acetic acid, glacial1mL
Ethanol, 90%100mL

Solution C

MaterialAmount
Azocarmine1g
Acetic acid, glacial1g
Distilled water100mL

Solution D

MaterialAmount
Aniline0.06mL
Ethanol, 90%100mL

Solution E

MaterialAmount
Phosphotungstic acid5g
Distilled water100mL

Solution F

MaterialAmount
Aniline blue0.5g
Orange G2g
Oxalic acid2g
Phosphotungstic acid0.05g
Distilled water100mL

Solution G

MaterialAmount
Acetic acid1mL
Distilled water100mL

Tissue Sample

The instructions specify fixation in see Baley’s fixative. Other mercuric-chrome fixatives would probably be satisfactory. The instructions also specify 3-4 µ celloidin sections which have been attached to a slide, and from which the celloidin has been removed. Possibly, similar paraffin sections would be satisfactory.

Baley’s fixative consists of:

MaterialAmount
Mercuric chloride3.4g
Potassium dichromate3.4g
Concentrated formalin (40%)25mL
Water225mL
Return to fixation instructions.

Protocol

  1. Attach sections to slides and remove celloidin, or dewax sections with xylene and bring to ethanol.
  2. Although not specified, a water rinse followed by the iodine thiosulphate sequence and a second water wash, to remove mercury pigment would be advantageous at this point.
  3. Wash with 70% ethanol.
  4. Place into solution A for 45 minutes.
  5. Place into solution B for 1-2 minutes.
  6. Place into solution C for 2 hours at 56°C.
  7. Wash with water.
  8. Place into solution D until nuclei are red and the cytoplasm is pink.
  9. Place into solution B for 1-2 minutes.
  10. Place into solution E for 4 hours
  11. Place into solution F for 4 hours until basophils are blue.
  12. Return to solution E for 3-5 minutes.
  13. Wash with water.
  14. Rinse with solution G.
  15. Wash with water.
  16. Dehydrate with absolute ethanol.
  17. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Acidophils  –  orange-red
  • Basophils  –  blue
  • Chromophobes  –  pale grey

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.
    Citing:
    Koneff, (1938)
    Stain Technology, v.13, pp.49
March 30, 2023
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Kostowiecki’s Trichrome for Muscle, Cartilage and Collagen

By Protocols, Stain Type, Trichrome Staining, Trichrome, One-Step
Protocol

Kostowiecki's Trichrome

for Muscle, Cartilage and Collagen

7
steps
5
materials
print

Materials

  • A red nuclear stain
  • Solution A
    MaterialAmount
    Aniline blue0.06g
    Orange G0.2g
    Phosphomolybdic acid1g
    Distilled water100mL

Preparation of Solution A

  1. Add both dyes to the water.
  2. Bring to the boil for three minutes.
  3. Add the phosphomolybdic acid while hot.
  4. Cool and filter.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. Most trichrome stains benefit from picric acid or mercuric chloride fixation. Formalin fixed tissues may benefit from secondary fixation of sections in Bouin’s fluid.


Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Stain nuclei with a red nuclear stain.
  3. Place into the staining solution until darkly stained, 30-120 minutes.
  4. Rinse with distilled water.
  5. Place in 95% ethanol for 1 minute.
  6. Dehydrate with absolute ethanol.
  7. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  red
  • Muscle  –  orange
  • Cartilage  –  blue
  • Collagen  –  blue-green

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.
    Citing:
    Kostowiecki, (1932)
    Zeitschrift für wissenschaftliche Mikroskopie und für mikroskopische Technik,
    v. 49, pp. 337
March 30, 2023
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Krajian’s Iron Hematoxylin

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

Krajian's Iron Hematoxylin

8
steps
6
materials
print

Materials

MaterialAmountFunction
Hematoxylin6 gDye
Ferric ammonium sulfate6 gMordant
Ferric chloride6 gMordant
Potassium iodide6 g–
95% ethanol50 mLSolvent
Distilled water50 mLSolvent

Compounding Procedure

  1. Dissolve the hematoxylin in the ethanol.
  2. Dissolve the other ingredients in the water.
  3. Combine the solutions.

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for 3 minutes.
  3. Rinse with tap water.
  4. Dip briefly in 1% hydrochloric acid in 70% ethanol.
  5. Wash well in running tap water to blue.
  6. Rinse with distilled water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  black
  • Background  –  as counterstain or unstained

Notes

  • Gray states that this solution is, “an excellent general purpose hematoxylin”.
  • The stability of the solution is not commented on, but it is likely not stable for long.
  • This method is from a technique for micro-organisms, so the staining time may need to be adjusted for sections.

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.
March 30, 2023
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Krutsay’s Alum & Iron Hematoxylin

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

Krutsay's Alum & Iron Hematoxylin

6
steps
6
materials
print

Krutsay’s Alum hematoxylin gives very highly selective staining of nuclei. It may also be easily converted into an iron hematoxylin for use as an acid resistant nuclear stain.

Materials

Alum hematoxylin

MaterialAmountFunction
Hematoxylin1 gDye
Potassium alum50 gMordant
Distilled water1 LSolvent
Hydrochloric acid5 mLAcidifier
Sodium iodate0.2 gOxidant

Iron conversion

MaterialAmountFunction
Iron alum2 gMordant
Hydrochloric acid0.5 mLAcidifer
Distilled water100 mLSolvent

Compounding Procedure

  1. Mix the reagents under Alum hematoxylin together.
  2. Bring to a boil
  3. Cool and filter.
  4. The solution may be used immediately.

Conversion to Iron Hematoxylin

  1. Mix the reagents under Iron conversion together, and dissolve.
  2. For use mix the following:
    • Alum hematoxylin solution – 25 mL
    • Iron conversion solution – 2 mL

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into either staining solution for 5 minutes.
  3. Rinse with water and blue.
  4. Rinse well with water.
  5. Counterstain if desired.
  6. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue (alum) or black (iron)
  • Background  –  as counterstain or unstained

Notes

  • This is a progressive solution and is highly selective for nuclei.
  • The iron Alum is (FeNH4(SO4)2.12 H2O)
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

Safety Note

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

References

  1. Humason, G. L.
    Animal Tissue Techniques.
    W. H. Freeman and Co., San Francisco, CA, USA
March 30, 2023
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Kleinenberg’s Alum Hematoxylin

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

Kleinenberg's Alum Hematoxylin

8
steps
5
materials
print

There are two alternate formulae for Kleinenberg’s Alum hematoxylin.

Materials

Formula I

Hematoxylin

MaterialAmountFunction
HematoxylinsaturatedDye
100% ethanol100 mLSolvent

Alum

MaterialAmountFunction
Ammonium alumsaturatedMordant
70% ethanol100 mLSolvent

Calcium

MaterialAmountFunction
Calcium chloridesaturatedMordant
70% ethanol100 mLSolvent

Compounding Procedure

  1. Prepare each of the solutions.
  2. For use, combine:
    1. Alum solution – 85 mL
    2. Calcium solution – 15 mL
    3. Hematoxylin solution – 1 mL

Formula II

Hematoxylin

MaterialAmountFunction
HematoxylinsaturatedDye
100% ethanol100 mLSolvent

Alum A

MaterialAmountFunction
Ammonium alumsaturatedMordant
Calcium chloridesaturatedMordant
70% ethanol100 mLSolvent

Alum B

MaterialAmountFunction
Potassium alumsaturatedMordant
70% ethanol88 mLSolvent
Alum A solution12 mL–

Compounding Procedure

  1. Prepare each of the solutions.
  2. For use, combine:
    1. Alum B solution – 100 mL
    2. Hematoxylin solution – 3 mL

Protocol

  1. Bring sections to water with xylene and ethanol.
  2. Place into the staining solution for an appropriate time.
  3. Rinse well with water.
  4. Differentiate with acid ethanol if necessary.
  5. Rinse with water and blue.
  6. Rinse well with water.
  7. Counterstain if desired.
  8. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei  –  blue
  • Background  –  as counterstain or unstained

Notes

  • Although not stated, the saturated hematoxylin solution should presumably be allowed to ripen.
  • Formula I uses 1 mL of the saturated hematoxylin solution, whereas formula II uses 3 mL, i.e. it is considerably stronger. This may affect the staining times.
  • Alum and calcium chloride combined give aluminum chloride hematoxylin.
  • The staining time should be determined by trial.
  • Acid ethanol is 0.5% – 1% hydrochloric acid in 70% ethanol.
  • Blueing is done with alkaline solutions such as hard tap water, Scott’s tap water substitute, 0.1% ammonia water, 1% aqueous sodium acetate, 0.5% aqueous lithium carbonate etc.

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.
    Citing:
    Cole, A. C., (1884).
    The methods of microscopical research. (formula I), and
    Böhm, A. & Opel, A., (1907).
    Manuel de technique microscopique, Ed. 4, (formula II).
March 30, 2023
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Kurnick Stain for Plasma Cells

By Intracytoplasmic Granules, Plasma Cells, Protocols, Stain Target
Protocol

Kurnick Stain

for Plasma Cells

5
steps
3
materials
print

Materials

Stock pyronin

MaterialAmount
Pyronin Y2g
Distilled water100mL

Stock methyl green

MaterialAmount
Methyl green2g
Distilled water100mL

Working solution

MaterialAmount
Stock pyronin12.5mL
Stock methyl green7.5mL
Distilled water30mL

Storing stock solutions

The pyronin stock solution should be extracted with chloroform ten times to ensure purity. The methyl green stock solution should be extracted a minimum of six times, until the chloroform is no longer colored with any crystal violet. Store both solutions over a layer of chloroform.

Tissue Sample

Most fixatives should be satisfactory if fixation time is not extended, but avoid potassium dichromate or picric acid. Overnight with a 10% formalin variant should be satisfactory.

Protocol

  1. Bring sections to distilled water via xylene and ethanol.
  2. Place into the staining solution for 6 minutes in a coplin jar.
  3. Blot gently.
  4. Dehydrate with n-butanol, two changes of 5 minutes each.
  5. Clear with xylene and mount with a resinous medium.

Expected Results

  • Nuclei – green to blue-green
  • Plasma cell cytoplasm, Nissl substance, nucleoli – red
  • Other cell cytoplasm – pink to unstained

Notes

  • As with other methyl green-pyronin methods used for demonstrating nucleic acids, in critical applications a control section should be treated with a 0.1% solution of ribonuclease for one hour at 37°C to remove RNA, and another treated with distilled water alone. Both control sections should be compared with an untreated but stained section.

Safety Note

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

References

  1. Disbrey, B. D., (1970)
    Histological laboratory methods. pp.187-188.
    E. & S. Livingstone, Edinburgh and London, UK.
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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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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