Gomori’s Silver Impregnation Method for Reticulin Fibers Argyrophilic method

Purpose

To demonstrate reticulin fibers.

Principle

The tissue is oxidized by potassium permanganate to enhance subsequent staining of reticulin fibers. The excess permanganate is bleached by the oxalic acid. Ferric ammonium sulfate acts as the sensitizer and is replaced by the silver from the silver diamine solution. Silver diamine is produced by adding ammonium hydroxide to an aqueous solution of silver nitrate. A precipitate of silver oxide is formed in the solution but dissolves as the addition of ammonium hydroxide continues. When the sections are exposed to the silver diamine the aldehyde groups present in the tissue will reduce the diamine silver to metallic silver, however, this precipitate is not visible until the sections are treated with formalin to further impregnate the sections. The sections are toned (impregnated component changes from brown to black) by gold chloride and the excess unreduced silver is removed with sodium thiosulfate.

Solutions

1% Potassium Permanganate: (Make 1 liter) oxidizer

  • Potassium Permanganate 1 g
  • Distilled water 100 ml

 

5% Oxalic Acid: bleach

  • Oxalic Acid 5 g
  • Distilled water 100 ml
  • Expiry: 2 months

 

2.5% Ferric Ammonium Sulfate (Iron III Ammonium Sulfate): sensitizer

  • Ferric Ammonium Sulfate 2.5g
  • Distilled water 100 ml

 

10% Formalin Solution: external reducer

  • 37-40% Formaldehyde 10 ml
  • Distilled water 90 ml
  • Expiry: 6 months. Make in fume hood

 

0.2% Gold Chloride (can be re-used): toner (from brown to black)

Prepare by diluting a 1% Gold Chloride solution (sodium tetrachloroaurate)

  • 1% gold chloride 10 ml
  • Distilled water 40 ml

1% gold chloride is purchased

 

5% Sodium Thiosulfate: removes unreduced silver

  • Sodium Thiosulfate 5 g
  • Distilled water 100 ml
  • Expiry: 6 months

 

Ammonical Silver Solution

Stock Solutions:

10% Silver Nitrate:

  • Silver nitrate 10 g
  • Distilled water 100 ml
  • Use acid cleaned glassware to make (p 25)
  • Store in fridge

3% Sodium hydroxide

  • Sodium hydroxide 3 g
  • Distilled water 100 ml

Ammonium Hydroxide (28%)

 

Working Solution (use acid cleaned glassware)

Add ammonium hydroxide drop-by-drop to10 ml of 10% silver nitrate in an Erlenmeyer flask while shaking the container continuously until the brown precipitate is just barely dissolved. Add 5 ml of 3% sodium hydroxide and cautiously re-dissolve the precipitate with ammonium hydroxide until only a faint cloudiness remains. If this step is carried too far and the cloudiness disappears, add 10% silver nitrate drop by drop until the solution is faintly cloudy. Dilute this solution to 50 mls with distilled water and filter into an acid cleaned coplin jar.

See safety precautions for used reagent in Note #3

 

Nuclear Fast Red (Kernechtrot)

Aluminum sulfate 25 gm

Distilled water 500 ml

Nuclear fast red 0.5 gm

  1. Dissolve aluminum sulfate in DH2O.
  2. Add nuclear fast red and dissolve with aid of heat.
  3. Cool, filter and add a crystal/grain of thymol. (Prevents bacterial growth)

Stable for 2 months.

Caution: Irritant avoid contact and inhalation

PROCEDURE

Note: Acid cleaned glassware and plastic forceps MUST be used for silver

  1. Deparaffinize sections and hydrate to water.
  2. Oxidize in 1% potassium permanganate for 1 minute. (Do not use if solution is milky).
  3. Wash in several changes of distilled water
  4. Bleach in 5% oxalic acid until color is removed.
  5. Wash in distilled water-3 changes
  6. Sensitize in 2.5% ferric ammonium sulfate for 1 min.
  7. Wash in several changes of distilled water
  8. Impregnate with ammonical silver working solution for 1 minute
  9. Rinse once in distilled water (critical step)-too much rinsing=poor impregnation, insufficient rinsing=”non-specific” precipitation
  10. Reduce in 10% formalin for 2 minutes
  11. Rinse in distilled water.
  12. Check under microscope for staining. If excessive, the method should be repeated from step 6 (this produces an effect like differentiation).
  13. Tone in 0.2% gold chloride for 10 minutes
  14. Rinse well in distilled water.
  15. Place slides in 5% sodium thiosulfate for 2 minutes
  16. Wash in tap then rinse in 2 changes of distilled water
  17. Counterstain with nuclear fast red for 5 minutes
  18. Wash well in tap water. If not washed well slides will be cloudy
  19. Dehydrate quickly, clear and mount.

Results

  • Reticulin fibers: black, should see a linear pattern. (If granular repeat)
  • Other elements according to the counterstain

notes

  1. All glassware must be clean – acid washed with acid-alcohol (1% HCL in 70% alcohol), then rinse well in distilled water.
  2. Silver solution should be made up just before use.
  3. Ammonical silver solutions decompose on evaporation to form explosive “fulminating silver”. Do not stir, invert gently. Store in the refrigerator in a brown bottle. Do not expose to sunlight. Inactivate ammonical silver by adding excessive dilute HCL, NaCL(9%) or javex.
  4. Use charged slides
  5. Use nonmetallic forceps (plastic) and scoops (tongue depressors)
  6. If chemically cleaned or plastic forceps are not used in silver or iron stains (Perl’s), there is a risk of contamination from previous stains that can cause a false positive reaction and/or producing background or non-specific staining.

Control

Liver

References

IWK. (2007). IWK Laboratory Histology Staining Manual, Special Stains.

Carson, F. L., & Cappellano, C. H. (2015). Histotechnology: A Self-Instructional Text (4th Ed.).

American Society of Clinical Pathologists Press.

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Gomori's Silver Impregnation Method for Reticulin Fibers Argyrophilic method Copyright © 2022 by NSCC is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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