Aim: To identify reticulin fibers in sections.

Principle: Reticulin fibers are treated with potassium permagnate to produce sensitized sites for silver deposition. Silver is in a form readily able to precipitate as metallic silver. Formalin, a reducing agent causes deposition of metallic silver
at pH 9.0.Excess silver is removed by sodium thiosulphate solution .Treatment with gold chloride produces permanent precipitate.

Control:  Normal liver.

Reagents:-

Acidified potassium permagnate

0.5% potassium permagnate                                    95ml
3% sulfuric acid                                                           5ml
Solution should be made fresh.

Silver nitrate solution

To 5ml of 10% aqueous silver nitrate, add strong ammonia drop by drop until the precipitate which has formed initially is dissolved. Add 5ml of 3% sodium hydroxide. Again add strong ammonia drop by drop till the precipitate is completely dissolved. Add distilled water to make it 50ml and keep it in a jar.

2% Oxalic acid

Oxalic acid                                          2gm
Distilled water                                   100ml

4% aqueous iron alum

Ferric ammonium sulphate               
Distilled water                                  100ml

10% Formalin:–

Formaldehyde                                  10ml
Distilled water                                  100ml

0.2% Gold chloride:-

Gold chloride                                   0.2%
Distilled water                                100ml
Store in refrigerator

2% Sodium thiosulphate:-

Sodiun thiosulphate                      2gm
Distilled water                               100ml

Neutral red (acidified):-

Neutral red                                      1gm
Distilled water                                100ml
Glacial acitic acid                          1ml
Dissolve the dye in distilled water. Add the acid, mix, filter and store.

Procedure:-

1. Deparaffinize and bring the sections to water.
2. Oxidize in acidified potassium permagnate for 3 minutes.
3. Rinse in distilled water.
4. Decolorize with 2% oxalic acid for 1 minute.
5. Rinse in distilled water.
6. Put iron alum for 10 minutes.
7. Rinse in distilled water.
8. Put ammonical silver solution for 10 seconds.
9. Rinse in distilled water.
10. Immediately reduce with formalin for 2 minutes.
11. Wash in running tap water for 2 minutes.
12. Tone in 0.2% gold chloride for 2 minutes.
13. Rinse in distilled water.
14. Fix in 2% thiosulphate for 2 minutes.
15. Wash in water for 2 minutes.
16. Counter-stain with neutral red for 2 minutes.
17. Dehydrate, clear in xylene and mount in DPX.

Result:-

Reticulin fibres                                  black
Nuclei                                                 red

Note:-
All glassware should be cleaned thoroughly.

Silver solution should be made fresh.
Step 12 should be omitted in liver sections.
Counter-stain may be omitted in trefine biopsies.