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See also: Tissue Stains: 49,
52, 55,
56, 69,
72, 81.
PRINCIPLE OF THE TEST.
To stain paraffinized tissue sections for elastic fibers,
required in the diagnosis of certain diseases encountered in
anatomic pathology, including temporal arteritis,
arterial wall disease, etc.
SPECIMEN REQUIRED.
All human tissue, excised at surgery, outpatient clinics,
and postmortems, fresh or in fixative, along with a filled-out
Tissue Examination Form
(U. S. Standard Form 515, USSF515).
Tissue is fixed in 10% buffered neutral formalin,
or in any other well-fixed tissue. Paraffin tissue sections,
cut at 6 micrometers.
REAGENTS, INSTRUMENTATION.
1. 10% alcoholic hematoxylin solution.
2. 10% ferric chloride solution.
3. Verhoeff's Iodine Solution:
Iodine........................................ 2.0 ml
Potassium iodine.............................. 4.0 ml
Distilled water............................... 100.0 ml
4. Mix the crystals of iodine and the crystals of iodine in a flask.
Shake vigorously. Then gradually add the distilled water, 20 ml at a time.
5. Verhoeff's Elastic Stain Working Solution.
Alcoholic hematoxylin, 10%.................... 25.0 ml
Alcohol, 100% ethyl........................... 25.0 ml
Ferric chloride, 10%.......................... 25.0 ml
6. Mix well, then add:
Verhoeff's iodine solution.................... 25.0 mL
7. 2% Ferric Chloride Differentiating Solution:
Ferric chloride, 10%......................... 2.0 gm
Distilled water............................... 100 ml
8. Van Gieson's Solution: 5% Sodium Thiosulfate (Hypo) Solution.
STEP-BY-STEP DESCRIPTION.
1. Deparaffinize and hydrate to distilled water.
2. Stain in Verhoeff's elastic stain working solution for 15 minutes.
3. Wash in lukewarm running tap water 20 minutes.
4. Place in distilled water.
5. Differentiate in 2% ferric chloride solution. Check microscopically.
Elastic fibers are black and sharply fined; the background is gray.
6. Place in 5% sodium thiosulfate for 1 minute.
7. Wash in tap water for 5 minutes.
8. Place in distilled water.
9. Counterstain in Van Gieson's stain for 1 minute.
10. Dehydrate rapidly through 95% ethyl alcohol (2 changes) and absolute
ethyl alcohol (2 changes); clear in 2 changes of xylene substitute.
11. Mount with resinous medium.
12. Results:
Elastic fibers.................. black
Nuclei.......................... black
Collagen.......................... red
Other tissue structures.......... yellow
13. Wipe the back of the slide. While wet with the 2% ferric chloride
differentiation solution, check under low powder. Elastic fibers in
arterial walls should be black and the arterial wall muscle, gray.
14. Do not leave in van Gieson solution for more than 1 minute.
The picric acid component decolorizes the elastic fibers.
15. Rinse rapidly in 95% ethyl alcohol,
to avoid decolorizing the van Gieson solution.
REFERENCES.
1, Jones ML.
Connective Tissues and Stains. Chapter 9, pp. 139-162.
In: Bancroft JD, Gamble M.
Theory and Practice of Histological Techniques. Fifth Edition.
Edinburgh: Churchill Livingstone. 2002;:139-162.
ISBN 0-443-06435-0, 796 pages.
2. Verhoeff FH.
Some new staining methods of wide applicability.
Including a rapid differential stain for elastic tissue.
JAMA. 1908;50:876.
3. Mallory FB.
Pathological Technique.
Philadelphia: WB Saunders; 1942;:170-171.
4. Prophet EB, Mills B, Arrington JB, Sobin LH, eds.
Laboratory Methods in Histotechnology.
Washington, DC: Armed Forces Institute of Pathology. 1992;:.
ISBN 1-881041-00-X, 278 pages.
5. Mikel UV, ed.
Advanced Laboratory Methods in Histology and Pathology.
Washington, DC: Armed Forces Institute of Pathology. 1994;:.
ISBN 1-881041-13-1, 254 pages.