Verhoeff's Elastic Stain
Mallory, Sheehan and Hrapchak in:Histotechnology - A
Self-Instructional Text ASCP Press. American Society of Clinical Pathologists,
- Elastic fiber techniques are used for
the demonstration of pathologic changes
in elastic fibers. These include atrophy of the elastic tissue, thinning
or loss that may result from arteriosclerotic
changes, and reduplication, breaks, or splitting that may result from other
vascular diseases. The techniques also may be used to demonstrate normal
elastic tissue, as in the identification of veins and arteries, and to
determine whether or not the blood vessels have been invaded by tumor.
- 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 to 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,
or 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 tissue has 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.
- Any well-fixed tissue may be used.
- Mechanical stirrer
- Coplin jars
- Erlenmeyer flasks
- graduated cylinders.
- Cut paraffin sections at 4 to 5µm.
- Quality Control
- Use a section of aorta embedded
on edge, or a cross section of a large artery.
- Lugol's Iodine
..... 10.0 g
- Potassium iodide ..... 20.0 g
- Distilled water .....
- Put the iodine and potassium iodide in a flask with 200
mL of the water. Stir on a mechanical stirrer until the iodine dissolves
and then add the remaining water.
- 100% Ferric Chloride
- Ferric chloride ..... 50.0 g
- Distilled water ..... 500.0 mL
- Store in the refrigerator.
- Verhoeff’s Elastic Stain
Prepare fresh each time and mix in order:
- Hematoxylin, 5% in 95% alcohol (may be kept as a stock
solution) ..... 30.0 mL
- Ferric chloride, 10% solution ..... 12.0 mL
- Lugol's iodine ..... 12.0 mL
- Van Gieson's Solution
- Acid fuchsin, 1% aqueous ..... 20.0 mL
- Picric acid, saturated solution (14 g/L) ..... 380.0 mL
- 5% Sodium Thiosulfate
(Sodium thiosulfate ..... 50.0 g + Distilled water .....
- Deparaffinize sections and hydrate to distilled water.
- Place sections in Verhoeff’s elastic tissue stain for
- Wash in two changes of distilled water.
- Differentiate sections microscopically in 2% ferric chloride until the
elastic fibers are distinct and the background is colorless to light gray.
If the sections are differentiated too far, restain.
- Rinse sections in distilled water.
- Place sections in sodium thiosulfate for I minute.
- Wash in running tap water for 5 minutes.
- Counterstain sections in van Gieson's stain for 1 minute.
in 95% alcohol.
- Dehydrate in absolute alcohol, clear in xylene,
and mount with synthetic resin.
- Elastic fibers..... blue-black to black
Nuclei ..... blue to black
Other tissue elements ..... yellow
- It is easy to overdifferentiate this stain. If the background is completely colorless, so that a clear
yellow counterstain is obtained, the section may be overdifferentiated.
It is probably better to err on the side of underdifferentiation.
- Overdifferentiated sections may be restained 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.
- It is not necessary to remove mercury deposits before
staining, as they will be removed by the staining solution.
- 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 staining solution, the reagents must be added in the order given, with 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
- 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.