RE: Verhoeff-Van Gieson stain

From:"Morken, Tim"

Here is one:

Elastic Tissue:  Verhoeff-Van Gieson Method

To demonstrate elastic fibers, sections are overstained in a
Hematoxylin-Iron-Ferric Chloride solution in a dye-lake reaction. The ferric
chloride and iron function primarily as mordants and also as oxidizers in
assisting conversion of hematoxylin dye to hematin. Iodine may act as a
trapping agent for the cationic dye lake, facilitating staining of elastic
fibers and retarding the extraction of stain from elastic fibers during
differentiation. Differentiation is accomplished using a dilute ferric
chloride solution to break the tissue-mordant-dye complex.
10% formalin fixation.  Cut paraffin sections at 3-5 microns.
Caution:  The following chemicals used in this procedure are hazardous:
Iodine	Iodine is a highly toxic and corrosive poison. Extreme caution
should be taken when handling this substance.
Acid Fuchsin	Poison. Do not breathe dust.
For more information on each chemical, refer to it's Material Safety Data
1.	Verhoeff's Iron Hematoxylin
1.	5% Alcoholic Hematoxylin
Hematoxylin (D#-46)	1 gm
Absolute alcohol	20 mL
If  hematoxylin will not go into solution with stirring, use gentle heat.
2.	10% Ferric Chloride
Ferric Chloride (AP#-265)	10 gm
Distilled water	100 mL
3.	Weigert's iodine
Iodine (AP# 365)	2 gm
Potassium Iodide (AP# 595)	4 gm
Distilled water	100 mL
Dissolve potassium iodide in approximately 20 mL of water, add iodine.  Let
iodine dissolve, then add total volume of water (80 mL).
	Verhoeff's Stain Solution
5% Alcoholoic Hematoxylin	20 mL
10% Ferric Chloride	8 mL
Weigert's Iodine	8 mL

Solution should be made up fresh.
Prepare working solution in order
1.	Pour the alocholic Hematoxylin into a graduated cylinder
2.	Add the Ferric Chloride, shake and mix
3.	Add the Wiegert's iodine, and stir well.
Solution should be jet black.

2.	2% Ferric Chloride
Either make up direct 2% aq. solution or use above Verhoeff's 10% Ferric
Chloride as astock solution.
Ferric Chloride, 10%	10 mL
Distilled Water	40 mL

3.	Van Gieson Solution
Picric Acid (AP# 555), Saturated aqueous solution (about 1.22%)	100.0 mL
Acid Fuchsin (D# 001), 1% aq. solution (C.I. 42685)	5.0 mL
Solution is stable for 6 months.
Filter before each use.

4.	5% Sodium Thiosulfate (Hypo) Solution
Sodium Thiosulfate (AP# 770) (Hypo)	5 gms
Distilled Water	100 mL

Formalin-fixed aorta, skin or kidney sections cut at 3-5 um.

1.	Deparaffinize and hydrate to distilled water.
2.	Stain 15-60 minutes (15-20 minutes usually ample) in Verhoeff's Iron
Hematoxylin. Tissue should be completely black.
3.	Rinse off excess stain in lukewarm running tap water for 20 minutes
and place in distilled water.
4.	Differentiate in 2% aq. solution ferric chloride.
Control differentiation under microscope by rinsing slides in water to
check.  As a guide for differentiation look for elastic fibers within
arteries and large veins.  Differentiate until the elastic fibers are black
and the background is colorless or very pale purplish gray.  If
differentiation is carried beyond the end-point, return slides to staining
5.	Wash slides in distilled water.
6.	Place in 5% Sodium thiosulfate for 1-2 minutes.
7.	Wash in running water 5-10 minutes.
8.	Counterstain in Van Gieson solution for 1 minute.
Do not leave in this solution for more than 1 minute.
The picric acid component decolorizes the elastic fibers.
9.	Dehydrate, clear and mount in permanant mounting media.

Elastic fibers 	Intensely blue - black to black.
Nuclei 	Blue black
Collagen 	Pink to Red
Other tissue elements 	Yellow

The period of counterstaing with Van Gieson's solution must not be prolonged
as the picric acid will act to further differntiate the elastic stain.
1.	Wells, G. G., Manual of Histologic Techniques, 3rd ed., University
of Tennessee, 1966., p 182.

2.	Sheehan, D.C., and Hrapchak, B.B.: Theory and Practice of
Histotechnology, Second Edition, 1980, C.V. Mosby Co., St. Louis,

-----Original Message-----
From: Cook, Angela []
Sent: Monday, August 26, 2002 11:06 AM
To: HistoNet (E-mail)
Subject: Verhoeff-Van Gieson stain

I am looking for a protocol for the Verhoeff-Van Gieson stain for elastin.
Is there anyone who has it that can email or fax it to me?  If not does
anyone have a journal reference where I could find it?

Angela Cook

Children's Hospital
Columbus, OH

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