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Robb-Smith’s Impregnation for Reticulin

Protocol

Robb-Smith's Impregnation

for Reticulin

20
steps
10
materials
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Materials

  • Silver nitrate, 5% aqu.
  • Silver nitrate, 10% aqu.
  • Sodium hydroxide, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88)
  • Ammonium hydroxide 10% aqu.
  • Potassium permanganate, 0.25% aqu.
  • Oxalic acid, 1.5% aqu.
  • Formalin, 37.5% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulphate, 5% aqu.

Preparation of Robb-Smith’s Ammoniacal Silver

  1. Place 28.5 mL of 10% aqueous silver nitrate in a flask.
  2. Add 0.6 mL of 10% aqueous sodium hydroxide
  3. Add strong ammonium hydroxide by drops until the precipitate just dissolves.
  4. The solution should have a faint opalescence.
  5. Dilute to 100 mL with distilled water.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed tissue mounted on slides are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended. The paraffin wax does not need to be removed.

Protocol

  1. Place sections with the paraffin wax intact in 10% ammonia for 15 minutes
  2. Wash with tap water.
  3. Oxidize with 0.25% potassium permanganate for 5 minutes.
  4. Wash with tap water.
  5. Place in oxalic acid until bleached.
  6. Wash with tap water.
  7. Place in 5% silver nitrate for 1 hour.
  8. Rinse with distilled water.
  9. Treat with ammoniacal silver solution for 15 minutes.
  10. Wash with distilled water.
  11. Place in 37.5% formalin for 3 minutes.
  12. Wash with tap water.
  13. Rinse with distilled water.
  14. Tone with 0.2% gold chloride solution for 3 minutes.
  15. Rinse with distilled water.
  16. Fix in 5% sodium thiosulphate for 1 minute.
  17. Wash well with running tap water.
  18. Dehydrate with ethanol.
  19. Remove paraffin wax and clear with a few changes of xylene.
  20. Mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  grey

Notes

  • The 10% ammonium hydroxide is made by diluting 1 part strong ammonium hydroxide with 9 parts distilled water.
  • The 37.5% formalin solution (15% formaldehyde) is made by diluting 37.5 mL strong formalin with 62.5 mL tap water.
  • Ensure that the strong ammonium hydroxide is fresh and full strength. Keep well stoppered when not in use. Pour sufficient for use into a beaker, then immediately restopper the bottle. Do not return unused solution to the stock 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.
  • 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.