Anatomical Pathology Special Stains
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Reticulin 

Aim of special stain:
Demonstrate both thick and thin reticular fibres within the tissue stained
Diagnostic applications:
Carcinomas, sarcomas, various disease states in liver, spleen and kidney
Material supplied for surveys:
Normal liver cut at 3µm
Recommended control:
Normal liver, spleen or placenta tissues

1. James Method

Fume hood must be used when making up Methenamine solution.
DO NOT DISCARD SILVER NITRATE DOWN THE SINK. Discard in the appropriate labelled waste bottle.

Reagents – commercial source unknown, unless specified:

1. Acidified permanganate
a) 0.25% potassium permanganate 25 mL
b) 0.25% sulphuric acid 25 mL
Mix equal parts immediately before use.
2. 5% oxalic acid
a) Oxalic acid 5 g
b) Distilled water 100 mL
3. 10% silver nitrate
a) Silver nitrate 10 g
b) Distilled water 100 mL
4. Ammoniacal silver solution
Add strong ammonia to 20 mL silver nitrate (10%) until the resulting precipitate just dissolves.
To this solution add 10% silver nitrate drop wise until the solution is just slightly opaque. Double the final volume with distilled water.
DO NOT FILTER.
DISCARD AFTER USE.
5. 5% formalin
a) Formalin 5.0 mL
b) Distilled water 100 mL
6. 5% sodium thiosulphate
a) Sodium thiosulphate 5.0 g
b) Distilled water 100 mL
7. 0.2% gold chloride (sodium salt, commercially available)

DO NOT TAKE SHORTCUTS

Coated or positively charged slides are recommended as sections are prone to floating off.

Method:

  1. Take sections to water.
  2. Oxidise in acidified permanganate solution for 2 minutes.
  3. Rinse in water.
  4. Decolourise with oxalic acid for 2 minutes.
  5. Rinse in 3 changes of distilled water.
  6. Treat with 10% silver nitrate for 3 minutes.
  7. Rinse in 3 changes of distilled water.
  8. Impregnate with Ammoniacal silver solution for 3 minutes.
  9. Rinse in 3 changes of distilled water.
  10. Reduce in 5% Formalin solution for 2 minutes.
  11. Rinse in 3 changes of distilled water.
  12. Tone in gold chloride for 1 minute.
  13. Wash in water.
  14. Fix in Sodium thiosulphate for 1 minute.
  15. Wash in water.
  16. Apply preferred counterstain (e.g., Nuclear Fast Red, Kernechtrot) for 2 minutes.
  17. Wash, dehydrate, clear and mount.

Results:

Reticulin fibres – black
Nuclei and Cytoplasm – red

2. Gordon and Sweets method

Fume hood must be used when making up Methenamine solution
DO NOT DISCARD SILVER NITRATE DOWN THE SINK. Discard in the appropriate labelled waste bottle.

Reagents – commercial source unknown, unless specified:

1. 0.5% Potassium Permanganate (commercial preparation – Australian Biostain)
2. 3% Sulphuric Acid (commercial preparation – Australian Biostain)
3. Acidified Potassium Permanganate
a) 0.5% Potassium Permanganate 9 mL
b) 3% Sulphuric Acid 1 mL
Add 3% sulphuric acid to potassium permanganate and mix well
4. 1% Oxalic Acid (commercial preparation – Australian Biostain)
5. 2% Ferric Ammonium Sulphate (Ferric Alum) – (commercial preparation – Amber Scientific)
6. 10% Silver nitrate (commercial preparation – Australian Biostain)
7. 3% Sodium Hydroxide (commercial preparation – Australian Biostain)
8. Ammonia Solution – (commercial preparation – Merck)
9. Wilder’s Silver Solution
a) 10% Silver Nitrate 10 mL
b) Ammonia Apply drop by drop
c) 3% Sodium hydroxide 10 mL
d) RO (reverse osmosis) water up to 100 mL
Reagent preparation:
  • To 10mL of 10% silver nitrate, add ammonia solution dropwise until the precipitate first formed is just dissolved.
  • Add 10mL of 3% Sodium hydroxide.
  • Redissolve the precipitate by adding ammonia solution dropwise until the solution is almost clear.
  • Make up to 100mL with RO water.
  • Filter through filter paper (commercially sourced, Whatman’s No 1) into a dark bottle and store at 4°C.
  • Care must be taken when titrating ammonia solution into the silver solution. Excess ammonia results in a great loss of sensitivity, giving weak or incomplete lack of reticular fibre impregnation.
10. Formal Scott’s Tap water
a) Formaldehyde 5 mL
b) RO water 50 mL
c) Volume to discard 12.5 mL
d) Scott’s Tap Water 12.5 mL
Add formaldehyde to RO water and mix.
Remove the volume to discard, add Scott’s Tap Water and mix well
11. 5% Sodium Thiosulphate – (commercial preparation – Australian Biostain)

Method:

  1. Bring solution to room temperature before use.
  2. Dewax and take sections to water.
  3. Oxidise with Acidified Potassium Permanganate for 3 minutes.
  4. Wash well with RO water.
  5. Bleach in 1% Oxalic Acid for 1 minute.
  6. Wash well with RO water.
  7. Sensitise in 2% Ammonium Iron III Sulphate (Ferric Alum) for 10 minutes.
  8. Wash well with RO water.
  9. Impregnate with Wilder’s Silver Solution for 1 minute.
  10. Wash well with RO water.
  11. Reduce in Formal Scott’s Tap Water for 2 minutes.
  12. Check microscopically – should be a cornflake brown colour.
  13. Wash well with RO water.
  14. Fix in 5% Sodium Thiosulphate for 2 minutes.
  15. Wash well with RO water.
  16. Dehydrate, clear and mount.

Results:

Reticulin fibres – black
Background – golden brown

3. Gomori method

Fume hood must be used when making up Methenamine solution – DO NOT DISCARD SILVER NITRATE DOWN THE SINK. Discard in the appropriate labelled waste bottle.

Reagents – commercial source unknown, unless specified:

1. a) 0.5% Celloidin Stock Solution
i) Celloidin 2.5 g
ii) Absolute Alcohol 250 mL
iii) Diethyl Ether 250 mL
Dissolve celloidin in absolute alcohol and ether.
Store in closed stoppered bottles to prevent evaporation.
b) Celloidin Working Solution
i) i. 0.5% Celloidin, stock solution 15 mL
ii) Absolute Alcohol 235 mL
Pour 0.5% celloidin stock solution into staining dish.
Add absolute alcohol.
2. 95% Alcohol
a) Absolute Alcohol 950 mL
b) Distilled Water 50 mL
3. 1% Potassium Permanganate
a) Potassium Permanganate 10 g
b) Distilled Water 1000 mL
4. 3% Sulphuric Acid
a) Sulphuric Acid 3 mL
b) Distilled Water 97 mL
5. Acidified Potassium Permanganate
a) 0.5% Potassium Permanganate 9.5 mL
b) 3% Sulphuric Acid 0.5 mL
6. 1% Oxalic Acid in 10% Hydrobromic Acid
a) Hydrobromic Acid 100 mL
b) Distilled Water 900 mL
c) Oxalic Acid 10 g
7. 2% Ferric Alum
a) Ammonium Iron (III) Sulphate-12-Dihydrate 20 g
b) Distilled Water 1000 mL
8. 8% Ammonium Nitrate
a) Ammonium Nitrate 40 g
b) Distilled Water 500 mL
9. 4% Sodium Hydroxide
a) Sodium Hydroxide 20 g
b) Distilled Water 500 mL
10. 10% Silver Nitrate
a) Silver Nitrate 10 g
b) Distilled Water 100 mL
11. Ammoniacal Silver Solution
a) 8% Ammonium Nitrate 7 mL
b) Distilled Water 35 mL
c) 4% Sodium Hydroxide 8 mL
d) 10% Silver Nitrate 4.2 mL
  • Prepare fresh in Coplin jar in above order.
12. 10% Buffered Formalin 
a) Sodium Phosphate, dihydrate (NaH2PO4.2H2O) 200 g
b) Disodium Phosphate, anhydrous (Na2HPO4) 324.5 g
c) Hot tap water 1500 mL
d) Formaldehyde, 37% concentrated 5000 mL
e) Water 50000 mL (up to)
Dissolve sodium dihydrogen orthophosphate dihydrate and disodium hydrogen orthophosphate in hot tap water.
Mix solution with formaldehyde and water.
13. 0.2% Formalin
a) Distilled Water 980 mL
b) 10% Buffered Formalin 20 mL
14. 1% Gold Chloride
a) Gold Chloride 1 g
b) Distilled Water 100 mL
15. 0.01% Gold Chloride
a) 1% Gold Chloride 5 mL
b) Distilled Water 495 mL
16. 5% Sodium Thiosulphate
a) Sodium Thiosulphate 5 g
b) Distilled Water 100 mL

Method:

  1. Deparaffinise sections in xylene then absolute alcohol.
  2. Leave slides in 0.5% celloidin for 2 minutes.
  3. Leave slides in 80oC oven until dry, 2 minutes.
  4. Place slides in 70% alcohol (to harden celloidin) for 1 minute.
  5. Wash slides in running water. Rinse in distilled water.
  6. Oxidise in acidified potassium permanangate for 5 minutes.
  7. Wash in distilled water.
  8. Bleach until white in 1% oxalic acid in 10% hydrobromic acid for 1 minute.
  9. Wash in tap water and then distilled water for 2 minutes.
  10. Mordant in 2% ferric alum for 10 minutes.
  11. Wash in tap water for 2 min. Rinse well in distilled water.
  12. Immerse in ammoniacal silver solution for 1 minute.
  13. Rinse front and back of the slide briefly in 95% alcohol.
  14. One dip in 95% alcohol, then one flick.
  15. Immerse in 2 changes of 0.2% neutral buffered formalin, clearing in 1st and leaving in 2nd change for 2 minutes in total.
    (NOTE: NO longer than 2 minutes in TOTAL)
  16. Rinse in distilled water.
  17. Tone in 0.01% gold chloride for 40 seconds.
  18. Rinse in distilled water.
  19. Fix in 5% sodium thiosulphate for 1 minute. (NOTE: NO longer than 1 minute.)
  20. Rinse in running tap water for 2 minutes.
  21. Dehydrate, clear and mount.

Results:

Reticular fibres – black
Nuclei – grey

Disclaimer:

These methods are intended as a guide only. Laboratories that wish to implement these methods should perform internal validation prior to use. The RCPAQAP does not make any claim or warranty for the accuracy or performance of these methods.

References

  1. 2021 RCPAQAP Technical General Survey 1.

  2. BD. Disbrey and J.H. Rack. James Method for Reticulin. Histological Laboratory Methods. Churchill Livingstone; 1970:214-6.

  3. James, KR. A Simple Silver Method for the Demonstration of Reticulin Fibres. The journal of medical laboratory technology. 1967 Jan; 24(1):49-51.

  4. JD. Bancroft and A. Stevens. Theory and Practice of Histological Techniques, 2nd edition. Churchill Livingstone, New York, 1982:42-144.

Last updated on March 04, 2025

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