FIXATION:        10% buffered neutral formalin.

TECHNIQUE:        Paraffin sections cut at 5 μm.

SOLUTIONS:

5% Alcoholic Hematoxylin

Hematoxylin crystals, C.I. 75290 ---------------------------------------------                5.0 gm

Alcohol, 80% -----------------------------------------------------------------                100.0 ml

10% Ferric Chloride

Ferric chloride (FeCl3 ∙ 6H20) -------------------------------------------------                10.0 gm

Distilled water -----------------------------------------------------------------                100.0 ml

Lugol’s Iodine Solution

Potassium iodide ----------------------------------------------------------------                4.0 gm

Iodine --------------------------------------------------------------------------                2.0 gm

Distilled water ------------------------------------------------------------------                100.0 ml

       Dissolve the potassium iodide in a little of the distilled water, add the

       iodine and dissolve.  Add the remainder of the distilled water.

Verhoeff’s Staining Solution

5% alcohol hematoxylin -------------------------------------------------------                20.0 ml

10% ferric chloride -------------------------------------------------------------                10.0 ml

Lugol’s iodine -------------------------------------------------------------------                10.0 ml

       Prepare fresh each time.

2% Ferric Chloride

10% Ferric Chloride (FeCl3 ∙ 6H20) ------------------------------------------------        10.0 ml

Distilled water ------------------------------------------------------------------                40.0 ml

       

Van Gieson’s Stain

Acid Fuchsin, C.I. 42685 ------------------------------------------------------                0.1 gm

Picric acid, saturated aqueous (approximately 1.4%) ---------------------------                100.0 ml

1. Deparaffinize and hydrate to distilled water.
2. Place in Verhoeff’s staining solution for 10 minutes.
3. Wash briefly in running tap water and rinse in two changes of distilled water.
4. Differentiated in 2% ferric chloride for 15 seconds. Stop the differentiation with two changes of distilled water. Check microscopically for black elastic fiber staining and gray background. Repeat 2% ferric chloride treatment and distilled water rinses as necessary for adequate elastic fiber demonstration. It is better to slightly under differentiate the tissue, because the subsequent Van Gieson counterstain will extract some of the elastic stain. Brain and heart tissue should be less differentiated with ferric chloride.
5. Rinse in two changes of distilled water.
6. Counterstain in Van Gieson's solution for 1 minute.
7. Dehydrate in three changes of 95% alcohol (in Coplin jars) and complete dehydration in four changes of absolute alcohol.
8. Clear in three or four changes of xylene.
9. Mount with synthetic resin.

Elastic fibers --------------------------------------------------------        black

Nuclei --------------------------------------------------------------        blue to black

Collagen ------------------------------------------------------------        red

Other tissue elements ----------------------------------------------        yellow

This is our most popular stain for elastic tissue. The only problem is that not every technologist will differntiate the slides the same way in Step #4. Therefore, there will be some variation in the quality of the staining results.

Mallory, F.B.:  Pathological Technique, New York, Hafner, 1968, pp. 170-171.

Sheehan, D.C. and Hrapchak, B.B.:  Theory and Practice of Histotechnology, St. Louis, The C.V. Mosby Co., 1980, pp. 196-197.