RE: [Histonet] reticulin procedure

From:Tony Henwood


Our procedure is as follows:

Silver Reticulin Stain

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	

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
 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

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.
Reticulin fibres 					Black
Collagen 						Brown
Use chemically clean glassware.
Step 10. Reducing solution - slides must be agitated constantly until entire
section goes brown.
 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

-----Original Message-----
From: Tapper, Sheila []
Sent: Thursday, 4 March 2004 11:12 PM
Subject: [Histonet] reticulin procedure

Does anyone perform a reticulin stain using an acidulated Potassium
permanganate?  I would be interested in your procedure!
Sheila Tapper
St. Luke's Hospital
Duluth, MN

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