[Histonet] reticulin procedure
Tony Henwood
AnthonyH <@t> chw.edu.au
Thu Mar 4 15:47:29 CST 2004
Sheila,
Our procedure is as follows:
Silver Reticulin Stain
Principle:
The mechanism of staining is:
a) Sensitisation of the tissue by an oxidising agent,
b) Deposition of the positively charged diamine silver ions,
c) Reduction of the silver ions,
d) Toning with gold chloride to increase contrast and clarity,
The following technique uses a volumetric rather than a titrated silver
diamine solution.
Oxidation (periodic acid and chromium trioxide can be used as well as
potassium permanganate) produces reducing groups in reticulin (aldehydes),
which are the groups subsequently, demonstrated by the sensitisation and
reduction steps in the technique. If non-acidified pot permanganate is used,
alkene groups in lipofuscins can also give rise to aldehyde groups and stain
black. These are blocked if the pH of potassium permanganate is under 1.5.
Fixation: 10% buffered formalin.
Microtomy: paraffin sections at 5um.
Controls: Liver
Reagents:
1. Acidified permanganate solution:
0.25% Potassium Permanganate 20ml
0.25% Sulphuric acid 20ml
2. 2% Oxalic Acid
3. 4% aqueous iron alum (ferric ammonium sulphate).
4. 8% Ammonium Nitrate (16g in 200ml) - Warning: Strong Oxidizer - see
MSDS
5. 4% sodium hydroxide (8g in 200ml) - Warning: Corrosive - see MSDS
6. 10% silver nitrate - Warning: Corrosive - see MSDS
7. Silver Diamine solution: prepare in the following order just prior
to use.
8% ammonium nitrate 7ml
Distilled water 35ml
4% sodium hydroxide 8ml
10% silver nitrate 3.8ml
8. Reducing Solution (10% formalin)
Warning: Toxic - see MSDS
Distilled water 90ml
40% Formaldehyde 10ml
9. 0.1% Gold Chloride
10. 2% Sodium Thiosulphate
11. Kernechtrot Counterstain
Procedure:
1. Bring sections to distilled water.
2. Acidified Potassium permanganate 5 min.
3. Running Tap water Wash
4. 2% oxalic acid 1 min
5. Running Tap water Wash
6. 4% Iron alum 5 min
7. Distilled water Rinse rapidly
8. Silver Diamine solution 6 min
9. Rinse in distilled water Quick dip
10. Reducing solution 2 min
11. Distilled water Wash
12. 0.2% Gold chloride 30-60 sec(Tone)
13. Distilled water Rinse
14. 2% Sodium Thiosulphate 1-2 min
15. Rinse in water 2 min
16. Counterstain in Kernechtrot 3 min
17. Rinse in water 2 min
18. Absolute alcohol x 2 Wash
19. Clear and mount.
Results:
Reticulin fibres Black
Collagen Brown
Notes:
Use chemically clean glassware.
Step 10. Reducing solution - slides must be agitated constantly until entire
section goes brown.
Reference:
1. Naoumenko,J.,Feigin,I., (1974) Stain Tech 49(3):153-155.
Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318
http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html
-----Original Message-----
From: Tapper, Sheila [mailto:STapper <@t> slhduluth.com]
Sent: Thursday, 4 March 2004 11:12 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] reticulin procedure
Does anyone perform a reticulin stain using an acidulated Potassium
permanganate? I would be interested in your procedure!
Thanks!
Sheila Tapper
St. Luke's Hospital
Duluth, MN
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