Prepared by
ROY ELLIS
IMVS Division of Pathology
The Queen Elizabeth Hospital
Woodville Road, Woodville, South Australia 5011
Principle
Reticulin fibres have little natural affinity for silver solutions so, they must be treated with potassium permanganate to produce sensitised sites on the fibres where silver deposition can be initiated. The silver is in a form readily able to precipitate as metallic silver (diamine silver solution)The Optimal pH for maximum uptake of silver ions is pH 9.0. A reducing agent, formalin, causes deposition of silver in the form of metal. Any excess silver in the unprecipitated state is removed by treating with hypo. Gold chloride treatment renders the preparation permanent and produces a neutral black colour of high intensity.
Technical Points
1. Make the silver solution fresh each time. Ammoniacal silver solutions can be explosive when allowed to dry. Immediately after use neutralise the silver solution with saturated sodium chloride and discard.
2. Clean glassware (used for preparing silver solution) with glassware cleaning solution. Wash thoroughly before use with tap water and use distilled water for the final rinse.
3. It is important not to over dissolve the precipitate at any stage as this will result in a decrease in sensitivity.
4. Omit step 12 for untoned retics. (ie liver specimens).
5. Omit step 16 for haematology trephines.
Method
1 Deparaffinise sections with xylene then take through alcohols to water.
2 Oxidise in acidified potassium permanganate for 3 minutes
3 Rinse in distilled water.
4 Decolourise with 2% oxalic acid for 1 min
5 Rinse in distilled water.
6 Mordant in 4% iron alum for 10 minutes
7 Rinse in distilled water.
8 Impregnate in ammoniacal silver solution for 11 seconds
9 Rinse quickly in distilled water.
10 Immediately reduce with 10% aqueous formalin for 2 minutes
11 Wash in running tap water for 2 minutes
12 Tone in 0.2% gold chloride (Not liver cores-see technical point 4) for 2 minutes
13 Rinse in distilled water.
14 Fix with 2% aqueous sodium thiosulphate (hypo) for 2 minutes
15 Wash in water for 2 minutes
16 Counterstain with neutral red (optional-see technical note 5) for 2 minutes
17 Dehydrate, clear and mount.
Results
- Reticulin fibres ............................Black
- Nuclei .......................................Red
Reagent Formulae
1. Acidified Potassium Permanganate XE "Potassium permanganate:acidified. 37-"
0.5% Potassium Permanganate ---------- 95ml
3% Sulphuric Acid -------------------------- 5ml
2. Silver solution
To 5 ml of 10% aqueous silver nitrate add strong ammonia drop by drop until the precipitate which first forms is just dissolved. Add 5 ml of 3% sodium hydroxide then add strong ammonia drop by drop until the resulting precipitate is almost completely dissolved. Make up to 50 ml with distilled water and pour into a clean coplin jar. NOTE: If too much ammonia is added there is a great loss of sensitivity.
3. 2% aqueous oxalic acid
Oxalic acid (Analytical) ------------------ 2 g
Distilled water --------------------------- 100 ml
4 4% aqueous iron alum
Iron alum (Ferric Ammonium Sulphate) ----- 4 g
Distilled water --------------------------------- 100 ml
5 10% formalin
Formaldehyde (Ajax, 37 – 40g/L) ----------- 10 ml
Distilled water --------------------------------- 90 ml
6 0.2% gold chloride
Gold chloride (Sodium tetrachloroaurate, BDH) ---- 0.2 g
Distilled water ----------------------------------------- 100 ml
Rinse the entire contents of bottle of gold chloride (sodium chloroaurate) into a clean glass bottle. Store in the refrigerator.
7 2% Sodium thiosulphate
Sodium thiosulphate --------------------------- 2 g
Distilled water ---------------------------------- 100 ml
8 Neutral Red Stain – acidified
neutral red (CI 50040) -------------------------- 1.0 g
distilled water ----------------------------------- 100.0 ml
gl acetic acid ------------------------------------- 1.0 ml
Dissolve the dye in the distilled water. Add the acid. Mix well. Filter into the reagent bottle. Keeps well.
References
1 Gordon, H and Sweet, H.H. 1936 A simple method for the silver impregnation of reticulin. American Journal of Pathology, V12, p545.
2 Lillie, R.D. 1977 H.J. Conn's Biological Stains, 9th edition Williams and Wilkins