Verhoeff's Elastic Stain

Mallory, Sheehan and Hrapchak in:Histotechnology - A Self-Instructional Text ASCP Press. American Society of Clinical Pathologists, Chicago 1990
Purpose
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.
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 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.
Fixative
Any well-fixed tissue may be used.
Equipment
  • Mechanical stirrer
  • Coplin jars
  • Erlenmeyer flasks
  • graduated cylinders.
Technique
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.
Reagents
  • Lugol's Iodine ..... 10.0 g
  • Potassium iodide ..... 20.0 g
  • Distilled water ..... 1,000.0 mL
  • 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 ..... 1,000.0 mL)
Procedure
  1. Deparaffinize sections and hydrate to distilled water.
  2. Place sections in Verhoeff’s elastic tissue stain for I hour.
  3. Wash in two changes of distilled water.
  4. 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.
  5. Rinse sections in distilled water.
  6. Place sections in sodium thiosulfate for I minute.
  7. Wash in running tap water for 5 minutes.
  8. Counterstain sections in van Gieson's stain for 1 minute.
  9. Differentiate in 95% alcohol.
  10. Dehydrate in absolute alcohol, clear in xylene, and mount with synthetic resin.
Results
Elastic fibers..... blue-black to black
Nuclei ..... blue to black
Collagen..... red
Other tissue elements ..... yellow
Notes
  1. 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.
  2. Overdifferentiated sections may be restained at any step provided they have not been treated with alcohol.
  3. Do not prolong staining with van Gieson's solution as picric acid also will differentiate the stain further.
  4. It is not necessary to remove mercury deposits before staining, as they will be removed by the staining solution.
  5. 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.
  6. 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.
  7. 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.
  8. 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.