It seems you answered your own question.  Check again, step 4.  Check each slide microscopically until you see black fibers on a gray background.  Then place the slide in water and continue with the other steps.  With the VVG,it's better to SLIGHTLY underdifferentiate because some more of the hematoxylin stain will come out in the Van Gieson's counterstain.  Always save your hematoxylin solution and use it to restain your slides if you accidentally take overdifferentiate them.

Hope this helps,

Teri Johnson
Managing Director Histology Facility
Stowers Institute for Medical Research
1000 E. 50th St.
Kansas City, Missouri  64110
tjj@stowers-institute.org




-----Original Message-----
From: NIDAL E MUVARAK [mailto:nmuvarak@facstaff.wisc.edu]
Sent: Monday, March 03, 2003 4:11 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 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