Category

Metal Impregnation, Silver

Laidlaw’s Impregnation for Reticulin on Bouin Fixed Tissue

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Laidlaw's Impregnation

for Reticulin on Bouin Fixed Tissue

20
steps
10
materials
print

Materials

  • Iodine, 1% in 95% ethanol
  • Sodium thiosulphate, 5% aqu.
  • Potassium permanganate, 0.5% aqu.
  • Oxalic acid, 5% aqu.
  • Formalin, 1% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Silver nitrate, 60% aqu.
  • Strong ammonium hydroxide (s.g. 0.88)
  • Lithium carbonate, saturated aqu.
  • Neutral red, 1% aqu.

Preparation of Ammoniacal Silver

  1. Place 16 mL of the 60% silver nitrate in a flask.
  2. Add 185 mL of saturated lithium carbonate.
  3. Allow the precipitate to settle and decant the supernatent.
  4. Wash the precipitate with water, allow to settle, and decant several times.
  5. Add 60 mL distilled water.
  6. Add drops of strong ammonium hydroxide until the precipitate just redissolves.
  7. Dilute to 100 mL with distilled water.

Tissue Sample

5 µ paraffin sections of Bouin’s fluid fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. The method was intended for staining reticulin in skin, but there is no reason it would not be satisfactory for other tissues.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Place in alcoholic iodine for 3 minutes.
  3. Rinse well with tap water.
  4. Bleach with sodium thiosulphate for 3 minutes.
  5. Oxidise with potassium permanganate for 3 minutes.
  6. Rinse well with tap water.
  7. Bleach in Oxalic acid for a 5 minutes.
  8. Rinse well with tap water.
  9. Rinse with distilled water.
  10. Treat with ammoniacal silver for 10 minutess.
  11. Rinse with distilled water.
  12. Reduce in formalin for 10 minute.
  13. Rinse well with tap water.
  14. Rinse with distilled water.
  15. Tone with 0.2% gold chloride for 10 minutes.
  16. Rinse with distilled water.
  17. Wash well with running tap water.
  18. Counterstain with neutral red for 1 minute.
  19. Rinse with tap water.
  20. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Note that this method does not say to fix the impregnation with sodium thiosulphate. If sections fade or fixing the impregnation is considered desirable, then Place in 5% sodium thiosulphate for 5 minutes after step 16.
  • Ensure that both the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • 1% formalin is made by diluting strong formalin 1:100 with tap water (1 mL strong formalin, 99 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

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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Jalowy’s Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Jalowy's Impregnation

for Reticulin

8
steps
4
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Ammonium hydroxide, 1% aqu.
  • Formalin, 10% aqu.

Preparation of Jalowy’s Ammoniacal Silver

  1. Place 20 mL of 10% silver nitrate in a flask and add 1 mL of 40% sodium hydroxide.
  2. Mix well, then filter. Wash the precipitate well with distilled water several times and decant.
  3. Add 20 mL distilled water to the precipitate and suspend.
  4. Add strong ammonium hydroxide by drops until the precipitate is just dissolved.
  5. Dilute to 100 mL with distilled water.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse with distilled water.
  3. Place in prewarmed ammoniacal silver solution for 5-30 minutes at 30°C.
  4. Rinse with distilled water.
  5. Rinse with 1% ammonium hydroxide.
  6. Place in 10% formalin for 2-10 minutes.
  7. Rinse well with tap water.
  8. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  brown

Notes

  • This method was recommended for collagen and reticulin fibres on formalin fixed skin.
  • Ensure that the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep the ammonium hydroxide well stoppered when not in use. Pour sufficient for use into a beaker, then immediately restopper the stock container. Do not return unused ammonium hydroxide to the stock bottle.
  • The instructions for this method do not specify to tone, fix, or counterstain. If fading or a lack of contrast make it desirable, toning with 0.1% yellow gold chloride until satisfactory, and/or fixing with 5% sodium thiosulphate, and/or counterstaining with 1% aqueous neutral red may be used.

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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Gordon & Sweets Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gordon & Sweets Impregnation

for Reticulin

19
steps
11
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.880).
  • Sodium hydroxide, 3% aqu.
  • Oxalic acid, 1% aqu.
  • Iron alum, 2.5% aqu.
  • Formalin, 10% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulphate, 3% aqu.
  • Neutral red, 1% aqu.
  • Mallory bleach
    MaterialAmount
    Potassium permanganate, 0.5% aqu.47.5mL
    Sulphuric acid, 3% aqueous2.5mL

Preparation of Ammoniacal Silver

  1. Place 5 mL of the 10% silver nitrate in a 100 mL flask.
  2. Using a pasteur pipette, add a drop of strong ammonium hydroxide then swirl the solution for a few seconds.
  3. A precipitate will form at first.
  4. Continue adding ammonium hydroxide drop by drop and swirling until the precipitate is just redissolved.
  5. Add 5 mL of 3% sodium hydroxide and mix well.
  6. A precipitate will again form.
  7. Add drops of ammonium hydroxide until the precipitate is just redissolved, leaving a faint opalescence.
  8. Dilute to 50 mL with distilled water.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with the Mallory bleach for 5 minutes.
  3. Bleach in Oxalic acid for a few minutes.
  4. Rinse with distilled water.
  5. Rinse well with tap water.
  6. Sensitise with iron alum solution for 15 minutes.
  7. Rinse well with distilled water.
  8. Treat with ammoniacal silver for 30 seconds.
  9. Rinse well with distilled water.
  10. Reduce in formalin for 1 minute.
  11. Rinse well with tap water.
  12. Rinse with distilled water.
  13. Tone with 0.2% gold chloride solution.
  14. Rinse with distilled water.
  15. Fix in sodium thiosulphate for 10 minutes.
  16. Wash well with running tap water.
  17. Counterstain with neutral red for 1 minute.
  18. Rinse with tap water.
  19. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Gordon & Sweets also suggested a variant that used one of Foot’s ammoniacal silver solutions. Apart from that, the method is the same as given above.

    Foot’s ammoniacal silver

    • To 10 mL of 1% silver nitrate, add 0.1 mL of 40% potassium hydroxide.
    • Add strong ammonium hydroxide by drops until the precipitate just redissolves.
    • Make up the volume to 100mL with distilled water.
  • Ensure that both the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • Most references to the Gordon & Sweets’ reticulin stain specify that the ammoniacal silver solution should be made with 10.2% aqueous silver nitrate and 3.1% aqueous sodium hydroxide. No explanation is given. The 10% and 3% solutions respectively, as given by Gray, work satisfactorily.
  • Iron alum is ferric ammonium sulphate. For routine formalin fixed tissue 15 minutes in the iron alum is usually sufficient. If necessary the time may be extended up to 2 hours.
  • 10% formalin is made by diluting strong formalin 1:10 with tap water (10 mL strong formalin, 90 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

Safety Note

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

References

  1. Drury, R A, and Wallington, E A, (1967).
    Carleton’s histological technique., Ed. 5.
    Oxford University Press, London, England.
  2. Culling, C F A, Allison, R T, Barr, W T, (1985).
    Cellular pathology technique., Ed. 4.
    Butterworths, London, England.
  3. 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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Gridley’s Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gridley's Impregnation

for Reticulin

17
steps
9
materials
print

Materials

  • Silver nitrate, 2% aqu.
  • Silver nitrate, 20% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Periodic acid, 0.5% aqu.
  • Formalin, 3% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.

Preparation of Da Fano’s Ammoniacal Silver

  1. Place 10 mL of 20% silver nitrate in a flask.
  2. Add 0.2 mL of 40% sodium hydroxide.
  3. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  4. Make up to 80 mL with distilled water.

Tissue Sample

6 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 0.5% periodic acid for 15 minutes.
  3. Rinse well with distilled water.
  4. Sensitise with 2% silver nitrate for 30 minutes.
  5. Rinse well with distilled water.
  6. Treat with da Fano’s ammoniacal silver for 15 minutes.
  7. Rinse with distilled water.
  8. Reduce in 3% formalin for 3 minutes.
  9. Rinse well with tap water.
  10. Rinse with distilled water.
  11. Tone with 0.5% gold chloride solution for 5 minutes.
  12. Rinse with distilled water.
  13. Fix in 5% sodium thiosulphate for 5 minutes.
  14. Wash well with running tap water.
  15. Counterstain with neutral red for 1 minute.
  16. Rinse with tap water.
  17. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Ensure that both the ammonium hydroxide and potassium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • 3% formalin is made by diluting strong formalin with tap water (3 mL strong formalin, 97 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Although the method specifies 5 minutes toning in 0.5% gold chloride, this is not mandatory. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
  • The use of periodic acid followed by an easily reducible silver solution is obviously similar to the Jones’ method for basement membranes and carbohydrates. Note, however, that Gridley’s method applies ammoniacal silver solution at room temperature after sensitising in aqueous silver nitrate at room temperature, then applies formalin as a reducing agent, i.e. it is an argyrophil reaction, while the Jones’ method applies a methenamine silver solution at 56°C for a longer time, without sensitising, and does not use an external reducer, i.e. it is an induced argentaffin reaction. Clearly, in Gridley’s method the periodic acid is being used for the same purpose as the potassium permanganate of the Mallory bleach in other methods.

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
Love0

Gluck’s Impregnation for Reticulin on Zenker Fixed Tissue

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Gluck's Impregnation

for Reticulin on Zenker Fixed Tissue

20
steps
12
materials
print

Materials

  • Oxalic acid, 5% aqu.
  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Formalin, 5% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Neutral red, 1% aqu.
  • Gram’s iodine.
    MaterialAmount
    Iodine1g
    Potassium iodide2g
    Distilled water300mL

    Mix the iodine and potassium iodide in a 500 mL flask. Add 5 mL of the water. When the iodine has dissolved, make up to 300 mL with distilled water.

  • Cajal’s Solution
    MaterialAmount
    Strong formalin15mL
    Ammonium bromide2g
    Water85mL

Preparation of Gluck’s Ammoniacal Silver

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 0.5 mL of 40% sodium hydroxide.
  3. Allow to settle, then decant the supernatent.
  4. Wash the precipitate, allow to settle, then decant a few times.
  5. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  6. Dilute to 100 mL with distilled water, then add 2 mL pyridine.

Tissue Sample

5 µ paraffin sections of Zenker fixed tissue are suitable. Other fixatives are likely to be satisfactory.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Rinse well with tap water.
  3. Place in Gram’s iodine for 4-8 hours.
  4. Rinsewith 70% ethanol.
  5. Rinse well with tap water.
  6. Bleach in 5% sodium thiosulphate for 5 minutes.
  7. Wash well with tap water.
  8. Place in Cajal’s solution at 37°C for 24 hours.
  9. Wash with tap water.
  10. Place in Gluck’s ammoniacal silver for 5 minutes.
  11. Rinse with distilled water.
  12. Place in 5% formalin for 5 minutes.
  13. Wash with tap water.
  14. Tone with 0.2% gold chloride solution until grey.
  15. Rinse with distilled water.
  16. Place in 5% sodium thiosulphate for 5 minutes.
  17. Wash well with tap water.
  18. Counterstain with neutral red for 1 minute.
  19. Rinse with tap water.
  20. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Ensure that both the ammonium hydroxide and sodium hydroxide are fresh and full strength. Keep both well stoppered when not in use. For the ammonium hydroxide, pour sufficient for use from the stock bottle into a beaker, then immediately restopper the stock bottle. Do not return excess ammonium hydroxide to the stock bottle.
  • After making the ammoniacal silver solution but before adding the pyridine, smell the solution to ensure it has only a faint smell of ammonia. If the smell of ammonia is strong it indicates that too much ammonium hydroxide has been added. If so, it is preferable to make the solution again. Improperly made ammoniacal silver solutions can affect the quality of the impregnation.
  • The formalin used to make Cajal’s solution should be neutralised, but do not use buffered formalin. Neutral formalin in this context may be made by keeping strong formalin over marble chips. However, be very careful as the gas given off may increase the pressure inside the container and cause an explosion. Either apply a cap loosely so gas can escape, or use a fermentation lock.
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

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
Love0

Foot’s Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Foot's Impregnation

for Reticulin

22
steps
22
materials
print

Materials

MaterialVariation IVariation IIVariation IIIVariation IV
Lugol’s iodine++++
Sodium thiosulphate, 1% aqu.++++
Sodium thiosulphate, 5% aqu.++++
Potassium permanganate, 0.25% aqu.++++
Oxalic acid, 5% aqu.++++
Strong ammonium hydroxide (s.g. 0.88)++++
Ammonium hydroxide, 0.1% aqu–+––
Foot’s tannin sol.–+––
Silver nitrate, aqu.2% & 10%1% & 10%10%10%
Formalin, aqu.5%20%20%1%
Yellow gold chloride, aqu.1%–0.2%0.2%
Foot’s gold sublimate–+––
Alkali, aqu.NaOH 40%KOH 40%LiC03 sat.Na2CO3
Weigert’s 1903 iron hematoxylin+–––
Harris alum hematoxylin––+–
van Gieson’s picro-fuchsin+–+–
Neutral red, 1% aqu.–––+

Lugol’s Iodine (see note)

MaterialAmount
Iodine6g
Potassium iodide4g
Distilled Water100mL

Mix the iodine and potassium iodide in a 200 mL flask. Add 10 mL of the water. When the iodine has dissolved make up to 100 mL with distilled water.

Foot’s gold sublimate

MaterialAmount
Yellow gold chloride0.2g
Mercuric chloride0.5g
Distilled Water100mL

Foot’s tannin solution

MaterialAmount
Tannic acid0.15g
Ammonium bromide3.5g
Strong formalin5mL
Distilled Water100mL

Preparation of Foot’s Ammoniacal Silver

Variation I

  1. Place 25 mL of 10% silver nitrate in a flask.
  2. Add 1 mL of 40% sodium hydroxide.
  3. Add ammonium hydroxide by drops until the precipitate is almost dissolved.
  4. Dilute to 100 mL with distilled water.

Variation II

  1. Place 10 mL of 1% silver nitrate in a flask.
  2. Add 0.1 mL of 40% potassium hydroxide.
  3. Add ammonium hydroxide by drops until the precipitate is almost dissolved.
  4. Dilute to 100 mL with distilled water.

Variation III

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 10 mL of saturated aqueous lithium carbonate.
  3. Let the precipitate settle, then decant the supernatent.
  4. Wash, allow to settle, and decant several times.
  5. Add 25 mL distilled water to the precipitate.
  6. Add ammonium hydroxide by drops until the precipitate is almost dissolved.
  7. Dilute to 100 mL with distilled water and filter.

Variation IV

  1. Place 10 mL of 10% silver nitrate in a flask.
  2. Add 40 mL of aqueous sodium carbonate (see note for concentration).
  3. Let the precipitate settle, then decant the supernatent.
  4. Wash, allow to settle and decant several times.
  5. Add ammonium hydroxide by drops until the precipitate is almost dissolved.

Tissue Sample

5 µ paraffin sections of Zenker fixed tissue are suitable. Other fixatives may be satisfactory. Carleton & Leach say that Var IV is suitable for 10% formalin, Zenker, Bouin or Helly fixed tissue. In all cases a section adhesive is recommended.

Protocol

StepVariation IVariation IIVariation IIIVariation IV
1Bring sections to water via xylene and ethanol (all variations).
2Place in Lugol’s iodine.5 min5 min5 min5 min
3Bleach in 1% sodium thiosulphate.30 sec30 sec30 sec30 sec
4Oxidise in 0.25% potassium permanganate.5 min5 min5 min5 min
5Bleach with 5% oxalic acid.10 min10 min10 min10 min
6Wash well with tap water.5 min5 min5 min5 min
7Rinse with distilled water.5 sec5 sec5 sec5 sec
8Place in 2% silver nitrate.48 hrs———
Place in Foot’s tannin solution at 37°C.—15 min——
Place in 0.1% ammonium hydroxide.—30 sec——
9Rinse with distilled water.5 sec5 sec——
10Place in Foot’s ammoniacal silver solution (Variation I).30 min———
Place in Foot’s ammoniacal silver solution (Variation II), 2 changes, each for:—5 min——
Place in Foot’s ammoniacal silver solution (Variation III) at 45°C.——15 min—
Place in Foot’s ammoniacal silver solution (Variation IV) at 45°C.———15 min
11Rinse with distilled water.5 sec5 sec5 sec—
12Reduce with 10% formalin.30 min———
Reduce with 20% formalin.—3 min2 min—
Flood with 1% formalin twice.———10 sec
13Rinse with distilled water.——5 sec—
Wash off with tap water.———5 sec
14Tone with 1% yellow gold chloride.1 hr———
Tone with Foot’s gold sublimate.3 min———
Tone with 0.2% yellow gold chloride.——2 min2 min
15Wash with distilled water.1 min1 min——
Wash with tap water.———2 min
16Place in 5% sodium thiosulphate.—3 min2 min2 min
17Wash with tap water.5 min5 min5 min5 min
18Place in Weigert’s iron hematoxylin.1 min———
Place in Harris’ alum hematoxylin.——3 min—
Place in neutral red.———1 min
19Wash with tap wate.5 min5 min5 min1 min
20Place in van Gieson’s picro-fuchsin.30 sec—45 sec—
22Dehydrate with ethanol, clear with xylene and mount in a resinous medium (all variations).

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  as counterstained
  • Background  –  grey

Notes

  • Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. After removing the amount required, immediately restopper the bottle.
  • Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
  • The formula for Lugol’s iodine may be an error. The amounts may have been reversed,
    i.e. It may be 4 g iodine and 6 g potassium iodide. Please refer to the page for
    Lugol’s iodine and compare Gray and Lee. The details for these methods are from Gray.
  • The instructions for ammoniacal silver solution Var IV says to add 40 mL of sodium carbonate to the silver nitrate solution. The sodium carbonate should be made as a 5% solution of the anhydrous salt or as an 11% solution of the dodecahydrate (12H2O).
  • 1%, 10% and 20% formalin solutions are made by diluting strong formalin appropriately with water, i.e.:
    • 1% is 1 mL strong formalin plus 99 mL water,
    • 10% is 10 mL strong formalin plus 90 mL water, and
    • 20% is 20 mL strong formalin plus 80 mL water.

Safety Note

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

References

  1. Gray, Peter. (1954), (for Var I, II & III)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.
  2. Carleton, H M, and Leach, E H, (1938), (for Var IV)
    Histological technique., Ed. 2.
    Oxford University Press, London, England.
March 30, 2023
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del Carpio’s Impregnation for Reticulin on Previously Stained Sections

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

del Carpio's Impregnation

for Reticulin on Previously Stained Sections

19
steps
15
materials
print

Materials

  • Potassium permanganate, 0.25% aqu.
  • Oxalic acid, 1% aqu.
  • Silver nitrate, 2% aqu.
  • Silver nitrate, 10% aqu.
  • Sodium hydroxide, 40% aqu.
  • Ammonia, 20% aqu. (see note)
  • Formalin, 10% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.
  • Ammoniacal silver

Solution Preparation

  1. Place 1 mL of 10% silver nitrate in a flask.
  2. Add 0.3 mL of 40% sodium hydroxide.
  3. While swirling, slowly add drops of 20% ammonia until the precipitate just redissolves.
  4. Make up to 100 mL with distilled water.

Tissue Sample

5 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. The method was designed to demonstrate reticulin in sections which have been previously stained with dyes.

Protocol

  1. Remove coverslip and bring slides to water with appropriate reagents.
  2. Oxidise with 0.25% potassium permanganate for 20 minutes.
  3. Rinse with tap water.
  4. Place in Oxalic acid until bleached.
  5. Wash with tap water.
  6. Sensitise with 2% silver nitrate 24 hrs.
  7. Rinse with distilled water.
  8. Treat with ammoniacal silver for 30 minutes.
  9. Rinse with distilled water, two changes.
  10. Reduce in 10% formalin for 15 secs.
  11. Rinse well with tap water.
  12. Rinse well with distilled water.
  13. Tone with 0.2% gold chloride solution.
  14. Rinse with distilled water.
  15. Fix in 5% sodium thiosulphate for 5 minutes.
  16. Wash well with running tap water.
  17. Counterstain with neutral red for 1 minute.
  18. Rinse with tap water.
  19. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey

Notes

  • Strong ammonium hydroxide (s.g. 0.88) contains about 30% ammonia by weight. The instructions for making the ammoniacal silver solution specify a 20% ammonia solution, so strong ammonium hydroxide should be diluted about 2:1 with distilled water. Since the final step is to make up to 100 mL with distilled water, the precise concentration is not too important.
  • 10% formalin is made by diluting strong formalin 1:10 with tap water (10 mL strong formalin, 90 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

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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Gomori’s Methenamine Silver for Glycogen and Fungi

By Aldehydes, Carbohydrates, Metal Impregnation, Metal Impregnation, Silver, Protocols, Stain Target, Stain Type
Protocol

Gomori's Methenamine Silver

for Glycogen and Fungi

15
steps
10
materials
print

This method is also known as Grocott’s or Grocott-Gomori’s methenamine silver.

Materials

  • Chromium trioxide, 5% aqu.
  • Neutral red, 1% aqu. or Light green SFy, 0.2% in 0.2% acetic acid, or Progressive hemalum and eosin
  • Sodium bisulfite, 1% aqu.
  • Sodium thiosulfate, 2% aqu.
  • Yellow gold chloride, 0.1% aqu.
  • Stock Methenamine silver
    MaterialAmount
    Methenamine, 3% aqu.100mL
    Silver nitrate, 5% aqu.5mL

    Shake until the precipitate redissolves. Silvering of the container indicates deterioration.

  • Working Methenamine silver
    MaterialAmount
    Stock Methenamine silver25mL
    Distilled water25mL
    Borax*0.1g

    Make just before use and preheat to 50°C.

    *Or 2 mL of a 5% aqueous borax solution (Grocott)

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 5% chromic acid (chromium trioxide) for 60-90 minutes.
  3. Rinse well with tap water.
  4. Bleach with sodium bisulphite for 1 minute.
  5. Rinse well with tap water.
  6. Rinse with distilled water.
  7. Treat with methenamine silver solution at 50&degC. until impregnated (up to 3 hours)
  8. Wash with distilled water.
  9. Tone with 0.1% gold chloride solution for 5 minutes.
  10. Rinse with distilled water.
  11. Fix in 2% sodium thiosulphate for 5 minutes.
  12. Wash well with running tap water.
  13. Counterstain with light green, neutral red or a light H&E.
  14. Rinse with tap water.
  15. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Oxidisable carbohydrates, including glycogen and fungi  –  black
  • Background  –  as counterstained

Notes

  • Methenamine is also known as hexamethylenetetramine and hexamine.
  • Borax is sodium tetraborate. Grocott’s modification adds the 0.1 g of borax as 2 mL of a 5% aqueous solution.
  • Aqueous solutions of chromium trioxide are usually referred to as chromic acid. Ten minutes in a 10% aqueous solution will usually give the same result as 60 minutes in a 5% solution.
  • Toning is a variable step. Untoned sections give dark brown material on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black material on a pale grey-brown background. Others tone longer (a few minutes) to produce black material on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
  • This method depends on a similar principle to Bauer’s chromic acid Schiff method, but in which the aldehydes produced by oxidation reduce a silver solution instead of combining with Schiff’s reagent to form a red compound. Consequently, those materials which are red in a Bauer’s stain will be black in Gomori’ stain, i.e. it is not specific for glycogen but will demonstrate any carbohydrates which can be oxidised to aldehydes, including fungi, and itis often used for that purpose.
  • In a similar method Hayashi, Tome and Shimosato recommended that thiosemicarbazide should be applied to the section after oxidation. Thiosemicarbazide has the formula H2NNHCSNH2. The hydrazine group (H2NNH-) combines with any aldehydes present. The thiocarbamyl group (-CSNH2) is a more powerful reducing agent than the aldehydes it replaces and reduces the methenamine silver solution more rapidly and with higher contrast.Immediately following step 5:
    • Place sections in 1% aqueous thiosemicarbazide for 10 minutes.
    • Wash well with tap water, and carry on from step 6.
  • It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide. The MSDS says:
    • Flash Point: n/a
    • Lower Explosive Limit: n/a
    • Upper Explosive Limit: n/a
    • Unusal Fire and Expl.rds: none identified

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, and Wallington, E A, (1967).
    Carleton’s histological technique., Ed. 5.
    Oxford University Press, London, England.
  3. Hayashi, I., Tome, Y. and Shimosato, Y., (1989)
    Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
    Stain Technology, v 64, p 185.
March 30, 2023
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Bensley & Bensley’s Impregnation for Reticulin

By Metal Impregnation, Metal Impregnation, Silver, Protocols, Reticulin, Stain Target, Stain Type
Protocol

Bensley & Bensley's Impregnation

for Reticulin

18
steps
14
materials
print

Materials

Solutions

MaterialVar IVar II
Lugol’s iodine++
Potassium permanganate, 1% aqu.++
Oxalic acid, 5% aqu.++
Sodium thiosulphate, 3% aqu.++
Tannic acid, saturated in ethanol, 95%.+–
Strong ammonium hydroxide (s.g. 0.88)++
Ammoniated water+–
Silver nitrate, aqu.1%2%
Formalin, 20% aqu.++
Yellow gold chloride, 0.2% aqu.++
Sodium hydroxide, 40% aqu.++
Neutral red, 1% aqu.++

Lugol’s iodine

MaterialAmount
Iodine1g
Potassium iodide2g
Distilled water300mL

Mix the iodine and potassium iodide in a 500 mL flask. Add 5 mL of the water. When the iodine has dissolved make up to 300 mL with distilled water.

Ammoniacal silver – Var I

Place 20 mL of 1% silver nitrate in a flask. Add 4 drops of 40% sodium hydroxide Add ammonium hydroxide by drops until the precipitate is almost dissolved. Dilute 1:10 with distilled water.

Ammoniacal silver – Var I

Place 20 mL of 2% silver nitrate in a flask. Add 3 drops of 40% sodium hydroxide Add ammonium hydroxide by drops until the precipitate is just dissolved.

Tissue Sample

Bensley & Bensley said that sections of tissue fixed in Zenker, Helly, ethanol or formalin are suitable. Although they commented that Var I gave a complete impregnation of paraffin or celloidin embedded tissue, they recommended Var II for paraffin sections because of their tendency to detach from slides. A section adhesive is recommended.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise in 1% potassium permanganate for 5 min.
  3. Bleach with 5% oxalic acid.
  4. Place in Lugol’s iodine.
  5. Bleach in 3% sodium thiosulphate.
  6. Wash with water.
  7. For Var I:
    1. Place in tannic acid solution at 56°C for 5 min.
    2. Rinse with ammoniated distilled water.

    For Var II:

    1. Place in 2% silver nitrate for 16 hrs.
  8. For Var I: Ammoniacal silver solution, VAR I, at 56°C for 10 min. For Var II: Ammoniacal silver solution, VAR II, at room temperature for 30 min.
  9. Wash with water.
  10. Reduce with 20% formalin for 3 min.
  11. Wash with water.
  12. Tone with 0.2% yellow gold chloride.
  13. Wash with water.
  14. Place in 3% sodium thiosulphate.
  15. Wash with water.
  16. Place in neutral red for 1 min.
  17. Wash with water.
  18. Dehydrate with ethanol, clear with xylene and mount in a resinous medium

Expected Results

  • Reticulin fibres – black
  • Nuclei – as counterstained
  • Background – grey or as counterstained

Notes

  • In the method details above, several steps do not have times given, meaning that the step is required but no other details were given. Common sense should prevail, and the step done for sufficient time to accomplish its obviously intended purpose. If it is a water wash for removal of an excess of the preceding material it would usually be for approximately 1-2 minutes. If it is for toning with gold chloride then see the final note below.
  • The method details also often specify to “Wash in water” without saying whether distilled or tap water should be used. In many cases it does not matter, but common sense should prevail. If tap water is likely to produce a non-specific precipitate of silver, then use distilled water and, when it specifies to “wash”, give several changes. Tap water varies in quality and individual laboratory’s results may differ due to that. Of course, distilled water could be used throughout, but it is strongly recommended after the silver or gold chloride solutions since these may be affected by tap water contaminants.
  • Bensley & Bensley said that “the silver carbonate solution of Hortega” could be substituted for their own silver oxide solution in Var I. Hortega gave form several such solutions and the authors do not say which one they meant. These formulas differ mainly by the amounts of 10% aqueous silver nitrate added to 5% aqueous sodium carbonate. All redissolve the resulting precipitate with drops of strong ammonium hydroxide.Hortega’s Ammoniacal silver solutions
    • Place 50 mL of 5% sodium carbonate in a flask. Add 12 mL of 10% silver nitrate. Let the precipitate settle, then decant the supernatent. Wash, allow to settle and decant several times. Add ammonium hydroxide by drops until the precipitate is almost dissolved. Dilute to 100 mL with distilled water.
    • In addition to the formula above, another adds 12.5 mL silver nitrate, does not decant and wash, but does dilute to 100 mL with distilled water.
    • A third adds 20 mL silver nitrate to 80 mL sodium carbonate, does not decant and wash, and does not dilute with distilled water.
    • A fourth adds 25 mL silver nitrate to 75 mL sodium carbonate, does not decant and wash, and does not dilute with distilled water.
    • A fifth adds 12 mL silver nitrate to 50 mL saturated aqueous lithium carbonate, decants and washes, and dilutes to 100 mL with distilled water.
  • Ensure that the ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. After removing the amount required immediately restopper the bottle.
  • Improperly made ammoniacal silver solutions can affect the quality of the impregnation. There should be a faint, persistent opalescence, with only a faint smell of ammonia.
  • 20% formalin is made by diluting 20 mL strong formalin with 80 mL water.
  • Bensley & Bensley suggested either Heidenhain’s Azan or 1% aqueous acridine red as counterstains. I have substituted neutral red.
  • The formula given for Lugol’s iodine is now usually referred to as Gram’s iodine.
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black reticulin fibres on a pale grey-brown background. Others tone longer (a few minutes) to produce black reticulin fibres on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.

Safety Note

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

References

  1. Bensley R. R. and Bensley, S. H., (1938)
    Handbook of Histological and Cytological Technique.
    U. Chicago Press, Chicago, USA
  2. 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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Jones’ Impregnation for Basement Membranes

By Aldehydes, Carbohydrates, Metal Impregnation, Metal Impregnation, Silver, Protocols, Stain Target, Stain Type
Protocol

Jones' Impregnation

for Basement Membranes

13
steps
9
materials
print

Materials

  • Periodic acid, 0.5% aqu.
  • Neutral red, 1% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Light green SFy, 0.2% in 0.2% acetic acid, or progressive hemalum and eosin
  • Sodium thiosulfate, 2.5% aqu.
  • Stock Methenamine silver
    MaterialAmount
    Methenamine, 3% aqu.100mL
    Silver nitrate, 5% aqu.5mL

    Shake until the precipitate redissolves. Silvering of the container indicates deterioration.

  • Working Methenamine silver
    MaterialAmount
    Stock Methenamine silver50mL
    Borax, 5% aqu.5mL

    Make just before use and preheat to 50°C.

Tissue Sample

3µ paraffin sections of neutral buffered formalin or Bouin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. Thinner sections are to be preferred. This method gives excellent results with deplasticized methyl methacrylate sections at 1µ.

Protocol

  1. Bring sections to water via xylene and ethanol.
  2. Oxidize with 0.5% periodic acid for 15 minutes.
  3. Rinse well with tap water.
  4. Rinse with distilled water.
  5. Treat with methenamine silver solution at 50&degC. until impregnated (up to 3 hours)
  6. Wash with distilled water.
  7. Tone with 0.2% gold chloride solution for 2 minutes.
  8. Rinse with distilled water.
  9. Fix in 2.5% sodium thiosulfate for 3 minutes.
  10. Wash well with running tap water.
  11. Counterstain with light green, neutral red or a light H&E.
  12. Rinse with tap water.
  13. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Basement membranes  –  black
  • Oxidisable carbohydrates  –  black
  • Background  –  as counterstained

Notes

  • In order to see the basement membranes on edge it is necessary to use the thinnest sections possible, especially for glomeruli.
  • Methenamine is also known as hexamethylenetetramine and hexamine.
  • Borax is sodium tetraborate.
  • Toning is a variable step. Untoned sections give dark brown material on a paler brown background. Many microscopists prefer to tone for about 15 seconds to produce brown-black material on a pale grey-brown background. Others tone longer (a few minutes) to produce black material on a grey background. Longer toning produces purple tones. Tone according to the personal preference of the microscopist reviewing the slides.
  • This method depends on a similar principle to the periodic acid Schiff reaction, but in which the aldehydes produced by oxidation reduce a silver solution instead of combining with Schiff’s reagent to form a red compound. Consequently, those materials which are red in a PAS will be black in Jones’ stain, i.e. it is not specific for basement membranes but will demonstrate any carbohydrates which can be oxidised to aldehydes.
  • Hayashi, Tome and Shimosato recommended that, after oxidation with periodic acid, thiosemicarbazide should be applied to the section. Thiosemicarbazide has the formula H2NNHCSNH2. The hydrazine group (H2NNH-) combines with any aldehydes generated by periodic acid oxidation. The thiocarbamyl group (-CSNH2) is a more powerful reducing agent than the aldehydes it replaces and reduces the methenamine silver solution more rapidly and with higher contrast.Immediately following step 3:
    • Place sections in 1% aqueous thiosemicarbazide for 10 minutes.
    • Wash well with tap water, and carry on from step 4.
  • It is well known that metallic azides can be explosive. However, thiosemicarbazide is not a simple metallic azide. The MSDS says:
    • Flash Point: n/a
    • Lower Explosive Limit: n/a
    • Upper Explosive Limit: n/a
    • Unusal Fire and Expl.rds: none identified

Safety Note

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

References

  1. Drury, R A, and Wallington, E A, (1967).
    Carleton’s histological technique., Ed. 5.
    Oxford University Press, London, England.
  2. Hayashi, I., Tome, Y. and Shimosato, Y., (1989)
    Thiosemicarbazide used after periodic acid makes methenamine silver staining of renal glomerular basement membranes faster and cleaner.
    Stain Technology, v 64, p 185.
March 30, 2023
Love0