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Laidlaw’s Impregnation for Reticulin on Bouin Fixed Tissue

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.