Long's Impregnation
for Reticulin in Zenker-Formol Fixed Tissue

Solutions

Impregnation
Potassium permanganate, 0.25% aqu.
Oxalic acid, 5% aqu.
Silver nitrate, 50% aqu.
Silver nitrate, 10% aqu.
Lithium carbonate, saturated aqu.
Strong ammonium hydroxide (s.g. 0.88)
Ammoniated water, 0.05% aqu.
Formalin, 1% aqu.
Yellow gold chloride, 0.2% aqu.
Sodium thiosulphate, 5% aqu.
Counterstain
Azocarmine, 0.1% in 1% acetic acid, aqu.
Phosphotungstic acid, 5% aqu.
Light green SFY, 1% in 1% acetic acid, aqu. (see note)
Acetic acid, 1% aqu.
iso-Propanol
Long's ammoniacal silver
Place 10 mL of 50% aqueous silver nitrate in a flask. Add by drops and while swirling, 100 mL of saturated aqueous lithium carbonate. Allow the precipitate to settle and decant the supernatent. Add distilled water to wash the precipitate and decant the supernatent five times. Add strong ammonium hydroxide by drops until the precipitate is almost dissolved. Dilute to 100 mL with distilled water. Heat in an oven at 50°C for 30 minutes in an open beaker. Cool to room temperature and filter.

Tissue sample
5µ paraffin sections of Zenker-Formol fixed tissue are suitable. Other fixatives are likely to be satisfactory. A section adhesive is recommended.

Method

  1. Bring sections to water via xylene and ethanol.
  2. Carry out the iodine-thiosulphate sequence to remove mercury pigment.
  3. Place in 0.25% potassium permanganate for 2 minutes.
  4. Wash with tap water for 5 minutes
  5. Bleach with 5% oxalic acid for 2 minutes.
  6. Wash thoroughly with tap water
  7. Rinse thoroughly with distilled water.
  8. Place in 10% silver nitrate for 12 hours in the dark.
  9. Wash with distilled water.
  10. Place in ammoniacal silver solution for 25-30 minutes at 40-45°C.
  11. Dip once in ammoniated water.
  12. Place in 1% formalin for 10-20 minutes.
  13. Wash with tap water.
  14. Rinse with distilled water.
  15. Tone with 0.2% gold chloride for 15-30 minutes.
  16. Rinse with distilled water.
  17. Fix in 5% sodium thiosulphate for 2 minutes.
  18. Wash well with running tap water.
  19. Place in azocarmine solution for 15-30 minutes.
  20. Rinse with distilled water.
  21. Place in phosphotungstic acid for 3-6 hours.
  22. Rinse with distilled water.
  23. Place in light green solution for 3 minutes.
  24. Rinse with distilled water.
  25. Rinse with 1% acetic acid until colours are satisfactory.
  26. Dehydrate with iso-propanol, clear with xylene and mount with a resinous medium.

Expected results

Notes

  1. 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.
  2. 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.
  3. Light green SF yellowish is used for adult tissue. This should be replaced with fast green FCF for fetal tissue.

 

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

Long M E. (1948)
Differentiation of myofibrillae, reticular and collagenous fibrils in vertebrates.
Stain Technol. v.2, p.69-75.

 


 

 

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