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Zimmerman’s Impregnation for Reticulin in Decalcified Tissue

Protocol

Zimmerman's Impregnation

for Reticulin in Decalcified Tissue

12
steps
7
materials
print

Materials

  • Silver nitrate, 10% aqu.
  • Silver nitrate, 3% aqu.
  • Strong ammonium hydroxide (s.g. 0.88)
  • Sodium hydroxide, 10% aqu.
  • Formalin, 10% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.

Preparation of Zimmerman’s Ammoniacal Silver

  1. Place 25 mL of 10% aqueous silver nitrate in a flask.
  2. Add 10% aqueous sodium hydroxide until no more precipitate forms (with a Pasteur pipette).
  3. Add strong ammonium hydroxide by drops until the precipitate just dissolves.
  4. Dilute to 100 mL with distilled water.

Tissue Sample

5µ paraffin sections of neutral buffered formalin fixed and acid decalcified 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. Place in 3% silver nitrate for 2 days.
  3. Wash with distilled water.
  4. Place in ammoniacal silver solution until sections are yellow-brown.
  5. Rinse with distilled water.
  6. Place in 10 formalin until sections are dark brown.
  7. Wash with distilled water.
  8. Tone with 0.5% gold chloride until sections are grey-black.
  9. Wash with distilled water.
  10. Fix in 5% sodium thiosulphate for 1 hour.
  11. Wash well with running tap water.
  12. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Background  –  grey

Notes

  • 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.
  • This method is a modification of Studricka’s technique using a shorter time in silver nitrate (step 2), and a more dilute ammoniacal silver solution (step 4).

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.