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Gridley’s Impregnation for Reticulin

Gridley's Impregnation

for Reticulin



  • Silver nitrate, 2% aqu.
  • Silver nitrate, 20% aqu.
  • Strong ammonium hydroxide (s.g. 0.88).
  • Sodium hydroxide, 40% aqu.
  • Periodic acid, 0.5% aqu.
  • Formalin, 3% aqu.
  • Yellow gold chloride, 0.5% aqu.
  • Sodium thiosulphate, 5% aqu.
  • Neutral red, 1% aqu.

Preparation of Da Fano’s Ammoniacal Silver

  1. Place 10 mL of 20% silver nitrate in a flask.
  2. Add 0.2 mL of 40% sodium hydroxide.
  3. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves.
  4. Make up to 80 mL with distilled water.

Tissue Sample

6 µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.


  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 0.5% periodic acid for 15 minutes.
  3. Rinse well with distilled water.
  4. Sensitise with 2% silver nitrate for 30 minutes.
  5. Rinse well with distilled water.
  6. Treat with da Fano’s ammoniacal silver for 15 minutes.
  7. Rinse with distilled water.
  8. Reduce in 3% formalin for 3 minutes.
  9. Rinse well with tap water.
  10. Rinse with distilled water.
  11. Tone with 0.5% gold chloride solution for 5 minutes.
  12. Rinse with distilled water.
  13. Fix in 5% sodium thiosulphate for 5 minutes.
  14. Wash well with running tap water.
  15. Counterstain with neutral red for 1 minute.
  16. Rinse with tap water.
  17. Dehydrate with ethanol, clear with xylene, and mount with a resinous medium.

Expected Results

  • Reticulin fibres  –  black
  • Nuclei  –  red
  • Background  –  grey


  • Ensure that both the ammonium hydroxide and potassium 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.
  • 3% formalin is made by diluting strong formalin with tap water (3 mL strong formalin, 97 mL tap water).
  • Toning is a variable step. Untoned sections give dark brown reticulin fibres on a paler brown background. Although the method specifies 5 minutes toning in 0.5% gold chloride, this is not mandatory. 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.
  • The use of periodic acid followed by an easily reducible silver solution is obviously similar to the Jones’ method for basement membranes and carbohydrates. Note, however, that Gridley’s method applies ammoniacal silver solution at room temperature after sensitising in aqueous silver nitrate at room temperature, then applies formalin as a reducing agent, i.e. it is an argyrophil reaction, while the Jones’ method applies a methenamine silver solution at 56°C for a longer time, without sensitising, and does not use an external reducer, i.e. it is an induced argentaffin reaction. Clearly, in Gridley’s method the periodic acid is being used for the same purpose as the potassium permanganate of the Mallory bleach in other methods.

Safety Note

Prior to handling any chemical, consult the Safety Data Sheet (SDS) for proper handling and safety precautions.


  1. Gray, Peter. (1954)
    The Microtomist’s Formulary and Guide.
    Originally published by: The Blakiston Co.
    Republished by: Robert E. Krieger Publishing Co.