Wilder's Impregnation
for Reticulin

Solutions

Silver nitrate, 2% aqu.
Strong ammonium hydroxide (s.g. 0.88)
Sodium hydroxide, 40% aqu.
Potassium permanganate, 1% aqu.
Oxalic acid, 5% aqu.
Uranium nitrate, 1% aqu.
Yellow gold chloride, 0.2% aqu.
Sodium thiosulphate, 5% aqu.
Neutral red, 1% aqu.
Developer
Strong formalin5 mL
Uranium nitrate0.15 g
Water1 L
Bielchowsky's ammoniacal silver
Place 48 mL of 2% silver nitrate in a flask. Add 0.4 mL of 40% sodium hydroxide. While swirling, slowly add drops of strong ammonium hydroxide until the precipitate just redissolves. Make up to 100 mL with distilled water.

Tissue sample
5µ paraffin sections of neutral buffered formalin fixed tissue are suitable. Other fixatives are likely to be satisfactory, but see the notes for comments about Zenker and Helly type fixed tissue. A section adhesive is recommended.

Method

  1. Bring sections to water via xylene and ethanol.
  2. Oxidise with 1% potassium permanganate for 1 minute.
  3. Rinse well with tap water.
  4. Bleach in Oxalic acid for a 1 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 Bielchowsky's ammoniacal silver for 1 minute.
  9. Rinse briefly with 90% ethanol.
  10. Place in the developer for 1 minutes.
  11. Rinse well with tap water.
  12. Rinse with distilled water.
  13. Tone with 0.2% gold chloride solution.
  14. Rinse with distilled water.
  15. Fix in 5% sodium thiosulphate for 1 minute.
  16. Wash well with running tap water.
  17. Counterstain with neutral red for 1 minute.
  18. Rinse with tap water.
  19. Dehydrate with ethanol, clear with xylene and mount with a resinous medium.

Expected results

Notes

  1. For Zenker and Helly type fixatives, Wilder recommended a slightly different procedure.
    1. Replace the Mallory bleach (steps 2-4) with 10% phosphomolybdic acid treatment for 1 minute, followed by a water rinse.
    2. Use Foot's ammoniacal silver solution instead of Bielchowsky's.
       
      Foot's ammoniacal silver
      To 10 mL of 1% silver nitrate in a flask add 0.1 mL of 40% potassium hydroxide. Add strong ammonium hydroxide drop by drop while shaking the solution until the precipitate just dissolves. Make up to 100 mL with distilled water.
  2. 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.
  3. 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.
  4. It is sometimes difficult to obtain uranium nitrate, particularly if it requires international transportation.
  5. The strong formalin used to make the developer should be neutralised, but do not use buffered formalin. Neutral formalin in this context may be made by keeping strong formalin over marble chips. However, be very careful as the gas given off may increase the pressure inside the container and cause an explosion. Either apply a cap loosely so gas can escape, or use a fermentation lock.
  6. 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.

 

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

 


 

 

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