RE: Verhoeff's Van Gieson Stain-Aldehyde Fuchsin

From:"Monson, Frederick C."

Hi Nidal,

I have a protocol for using Gomori's Aldehyde Fuchsin (AF) with H&E to
insure an elegant stain of elastin in tissues in which your primary interest
is NOT in the epithelium - the AF does tend to stain GAG secretors which
confuses the issue.  If I have to look/image GAG-secreting epithelium as
well as elastin, I would use Weigert's.  I have not tried to block the GAG's
which might be an option.

My methods are similar in that either the AF or Weigert's is applied before
the H&E which is applied in normal fashion - I have not found the acid
differentiation of hematoxylin to adversely affect the AF or Weigert's.

An example of the AF H&E was to be found on the listserver's image page, but
I cannot check tonight as the site appears to be down.


Fred Monson

Frederick C. Monson, PhD
Center for Advanced Scientific Imaging

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-----Original Message-----
Sent: Monday, March 03, 2003 5:11 PM
To: HistoNet Server
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
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
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
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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