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Verhoeff-Van Gieson (VVG) Staining Protocol for Elastic Fibers
Description: This method is used for identifying elastic fibers in tissues such as skin, aorta, etc. on formalin-fixed, paraffin-embedded sections, and may be used for frozen sections as well. The elastic fibers will be stained blue-black and background will be stained yellow.
Fixation:
10%
formalin.
Section:
paraffin sections at 5 um.
Solutions and Reagents:
5%
alcoholic hematoxylin:
Hematoxylin ---------------------------------- 5 g
100%
alcohol ---------------------------------- 100 ml
Mix
to dissolve with the aid of gentle heat. Filter.
10% aqueous
ferric chloride (prepare fresh, not necessary):
Ferric chloride -------------------------------- 10 g
Distilled water -------------------------------- 100 ml
Weigert’s iodine
solution:
Potassium iodide ------------------------------ 2 g
Iodine ------------------------------------------- 1 g
Distilled water --------------------------------- 100 ml
Use 4 ml of
distilled water to dissolve potassium iodide. And then add iodine.
Once iodine is dissolved, dilute this solution by adding 96 ml of
distilled water. This solution may be prepared fresh as needed or
made in larger quantities and stored in brown bottle in the dark at
room temperature.
Verhoeff’s
Working Solution:
The working staining solution should be made up fresh for best
results. It will not stain
satisfactorily if it is kept more than one working day.
Prepare the working solution by adding in order the following
reagents:
5%
alcoholic hematoxylin -------------------- 20 ml
10%
ferric chloride ---------------------------- 8 ml
Weigert’s iodine solution ------------------- 8 ml
Mix the above
amounts (or needed proportions thereof) well. Solution should be jet
black. Use immediately and discard after use.
2% aqueous ferric chloride (prepare fresh, not necessary):
10% ferric chloride from above
------------ 10 ml
Distilled water ------------------------------- 50 ml
5% aqueous sodium thiosulfate:
Van Gieson’s counterstain:
1% aqueous acid fuchsin
--------------------- 5 ml
Saturated aqueous picric acid
-------------- 100 ml
For nervous tissues may be prepared as
follows:
1%
aqueous acid fuchsin
------------------- 15 ml
Saturated aqueous picric acid ------------- 50 ml
Distilled water ------------------------------- 50 ml
Procedure:
1.
Deparaffinize and hydrate slides to distilled water.
2.
Stain
in Verhoeff’s solution for 1 hour. Tissue should be completely
black.
3.
Rinse
in tap water with 2-3 changes.
4.
Differentiate in 2% ferric chloride for 1-2 minutes.
5.
Stop
differentiation with several changes of tap water and check
microscopically for black elastic fiber staining and gray
background. It is better to slightly under differentiate the tissue,
since the subsequent Van Gieson’s counterstain can extract the
elastic stain somewhat.
6.
Wash
slides in tap water.
7.
Treat
with 5% sodium thiosulfate for 1 minute. Discard solution.
8.
Wash
in running tap water for 5 minutes.
9.
Counterstain in Van Gieson’s solution for 3-5 minutes.
10.
Dehydrate quickly through 95% alcohol,
2 changes of 100% alcohol.
11.
Clear
in 2 changes of xylene for 3 minutes each.
12.
Coverslip with resinous mounting medium.
Results:
Elastic fibers
--------------------- blue-black to black
Nuclei ----------------------------- blue to black
Collagen -------------------------- red
Other
tissue elements ---------- yellow
Positive Controls:
Aorta, Kidney, Myometrium