Hi Nidal,
There is no set time for the differentiation step.  I always would dip each
slide individually until the blackness color of the tissue would become a
light gray color.  The agitation of the ferric chloride  with the dipping
motion is the key.  Sometimes the black would fade right away, sometimes it
seemed it took forever, but do the slides individually and dip the slides by
hand.  See if that works.
Nancy



Nancy Weber
Research Associate
Veterinary Clinical Sciences
Ohio State University
----- Original Message -----
From: "NIDAL E MUVARAK" 
To: "HistoNet Server" 
Sent: Monday, March 03, 2003 5:11 PM
Subject: Verhoeff's Van Gieson Stain


> Hi everyone. I just started doing VVG stains for mouse lung and artery
tissues. I did two trial stains. It seems that the length of the step that
requires differentiation in 2% ferric chloride is pretty crucial. In the
first experiment I incubated the slides in 2% ferric chloride for about 30
sec. The result was horrible staining.... the tissue was pitch black, with
lots of junk in the background. In the second experiment, I incubated the
slides for a little over 5 min. That resulted in the elimination of all
nuclei and elastic fiber staining (which should be black according to the
protocol). So I was wondering what's the optimal time for differentiation
with 2% FeCl3. I will list the protocol I'm using below. If anyone sees
anything wrong with it, please let me know. Thank you for your time.
>
>
> VVG Stain:
> 1) Fix cryosections in cold aceton for 10 min. Dry. Rehydrate with
distilled water.
> 2) Incubate in Verhoeff's hematoxylin (20ml alcoholic hematoxylin, 8ml 10%
FeCl3, 8ml Lugol's iodine) for 30 min.
> 3) Wash in tap water
> 4)Differentiate in 2% FeCl3. Check microscopically for black fibers on
gray background.
> 5) Rinse in water
> 6) Rinse in 5% hypo solution for 1 min to remove iodine.
> 7)Wash in water.
> 8)Conterstain in Van Gieson's solution (1ml of 1% Acid Fuchin; 45ml of
saturated picric acid) for 5 min.
> 9)Rinse quickly with acidified water (5ml acetic acid in 1L distilled
water).
> 10) Dehydrate 3X 95% EtOH, 2X 100% EtOH, 3X xylene. Cover with resinous
mounting medium.
>
> Nidal E Muvarak
> Associate Research Specialist
> Department of Biomedical Engineering
> University of Wisconsin-Madison
> 1550 Engineering Dr.; Rm. 2158
> Madison, WI 53706-1609
> Lab: (608) 265-8921; Office: (608) 265-4205;
> Home: (608) 256-7934; Cell: (608) 332-6068
>
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