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Lillie’s Impregnation for Reticulin

Protocol

Lillie's Impregnation

for Reticulin

20
steps
8
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Potassium permanganate, 0.5% aqu.
  • Oxalic acid, 5% aqu.
  • Uranium nitrate, 1% aqu.
  • Formalin, 10% aqu.
  • Yellow gold chloride, 0.2% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.

Preparation of Lillie’s Ammoniacal Silver

  1. Place 20 mL strong ammonium hydroxide in a flask.
  2. Add 10% silver nitrate drop by drop until the precipitate is almost dissolved (about 25-30 mL). The solution should have a faint opalescence.
  3. Dilute to twice its volume 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 0.5% potassium permanganate for 2 minutes.
  3. Rinse well with tap water.
  4. Bleach in Oxalic acid for a 2 minutes.
  5. Rinse well with tap water.
  6. Sensitise with 1% uranium nitrate solution for 5-10 seconds.
  7. Rinse with distilled water.
  8. Treat with ammoniacal silver solution for 3 minute.
  9. Rinse briefly with 90% ethanol.
  10. Rinse with distilled water.
  11. Place in 10% formalin for 2 minutes.
  12. Rinse well with tap water.
  13. Rinse with distilled water.
  14. Tone with 0.2% gold chloride solution.
  15. Rinse with distilled water.
  16. Fix in 5% sodium thiosulphate for 2 minute.
  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

  • 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.
  • It is sometimes difficult to obtain uranium nitrate, particularly if it requires international transportation.
  • 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.