Verhoeff-van Gieson Method for Elastic Fibers
Purpose
To demonstrate elastic fibers
Principle
The tissue is overstained with a soluble lake of hematoxylin-ferric chloride-iodine. Both ferric chloride and iodine serve as mordants, but they also have an oxidizing function that assists in converting hematoxylin into hematein. The mechanism of dye binding is probably by formation of hydrogen bonds, but the exact chemical groups reacting with the hematoxylin have not been identified.
This method requires that the sections be overstained and then differentiated, so it is regressive. Differentiation is accomplished by using excess mordant (in this case, ferric chloride) to break the tissue-mordant-dye complex. The dye will be attracted to the larger amount of mordant in the differentiating solution and will be removed from the tissue. The elastic fibers have the strongest affinity for the iron-hematoxylin complex and will retain the dye longer than the other tissue elements. This allows other elements to be decolorized and the elastic fibers to remain stained.
Sodium thiosulfate is used to remove excess iodine. van Gieson’s solution is the most commonly used counterstain, but others may be used.
Solutions
Lugol’s Iodine
- Potassium iodide 2 g
- Iodine 1 g
- Distilled water 100 mL
Important: mix reagents in the order listed. Dissolve the potassium iodide in a flask with 20 mL of the water, then dissolve the iodine in this solution and add remaining water. Store in a brown bottle.
Expiry: 2 months
10% Ferric Chloride
- Ferric chloride 50 g
- Distilled water 500 mL
Store in the refrigerator.
Expiry: 6 months
2% Ferric Chloride
- Ferric chloride 2 g
- Distilled water 100 mL
Expiry: 1 month
5% Alcoholic Hematoxylin
- Hematoxylin 5 g
- Absolute alcohol 100 mL
Expiry: 4 months
Verhoeff’s Elastic Stain (Working Solution)
Prepare fresh each time and mix in the following order:
- 5% alcoholic hematoxylin 30 mL
- 10% ferric chloride 12 mL
- Lugol’s iodine 12 mL (helps form insoluble lake in the elastic fiber)
van Gieson’s Solution (1% acid fuchsin and saturated picric acid)
- 1% aqueous acid fuchsin 9 mL
- Saturated picric acid 50 mL
- Distilled water 50 mL
Can be purchased
SAFETY: Picric acid can become explosive when dry
5% Sodium Thiosulfate
- Sodium thiosulfate 5 g
- Distilled water 100 mL
Expiry 6 months
METHOD
- Deparaffinize sections and hydrate to distilled water.
- Place sections in Verhoeff’s elastic stain for 15-60 minutes (check with instructor for times to start with)
- Rinse in distilled water.
- Differentiate sections (regressive stain) in 2% ferric chloride for 1 minute and then check microscopically. The elastic fibers and nuclei should be black, and background should appear ‘dirty’. If the sections are over-differentiated, re-immerse in staining solution.
- Wash well in tap water.
- Place sections in 5% sodium thiosulfate for 1-2 minutes (removed excess iodine).
- Wash in running tap water for 5 minutes.
- Counterstain sections in van Gieson’s solution for 1 minute.
- Dehydrate rapidly (3 good dips in each alcohol), clear and mount.
results
Elastic fibers: black
Nuclei: brown/black
Collagen: red
Other tissue elements: yellow
Notes
- It is easy to over-differentiate this stain. If the background is completely colorless, so that a clear yellow counterstain is obtained, the section may be over-differentiated. It is probably better to err on the side of under-differentiation.
- Over-differentiated sections may be re-stained at any step provided they have not been treated with alcohol.
- Do not prolong staining with van Gieson’s solution as picric acid also will differentiate the stain further.
- The preparation of van Gieson’s solution is critical for proper differentiation of muscle and collagen. If the picric acid is not saturated, collagen will not stain red, and cytoplasm, muscle, and collagen may all stain the same color.
- To prepare the Verhoeff’s elastic working solution, the reagents must be added in the order given, mixing after each addition, or poor staining may result.
- The staining jar that contained the Verhoeff’s solution may be cleaned easily by transferring the 2% ferric chloride to the jar for a few minutes before discarding the solution.
- For optimum results, slides must be individually differentiated, as the time of differentiation is somewhat dependent on the amount of elastic tissue present. Do not depend on the control for timing the differentiation of all sections.
- It is not necessary to remove mercury pigment as this will be dissolved by the stain.
- It is a rapid method, but fails to demonstrate fine elastic fibers.
Control
Skin, heart, lung, or use a section of aorta embedded on edge, or a cross section of a large artery.
References
IWK. (2007). IWK Laboratory Histology Staining Manual, Special Stains.
Carson, F. L., & Cappellano, C. H. (2015). Histotechnology: A Self-Instructional Text (4th ed.). American Society of Clinical Pathologists Press.