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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