Description: This method is used for identifying elastic fibers in tissues such as skin, aorta, etc. on formalin-fixed, paraffin-embedded sections, and may be used for frozen sections as well. The elastic fibers will be stained blue-black and background will be stained yellow.

Fixation: 10% formalin.

Section: paraffin sections at 5 um.

Solutions and Reagents:

5%  alcoholic hematoxylin:

      Hematoxylin ---------------------------------- 5 g

      100% alcohol ---------------------------------- 100 ml

      Mix to dissolve with the aid of gentle heat. Filter.

10% aqueous ferric chloride (prepare fresh, not necessary):

      Ferric chloride -------------------------------- 10 g

      Distilled water -------------------------------- 100 ml

Weigert’s iodine solution:

      Potassium iodide ------------------------------ 2 g

      Iodine ------------------------------------------- 1 g

      Distilled water --------------------------------- 100 ml

      Use 4 ml of distilled water to dissolve potassium iodide. And then add iodine. Once iodine is dissolved, dilute this solution by adding 96 ml of distilled water. This solution may be prepared fresh as needed or made in larger quantities and stored in brown bottle in the dark at room temperature.

Verhoeff’s Working Solution:

The working staining solution should be made up fresh for best results. It will not stain  satisfactorily if it is kept more than one working day. Prepare the working solution by adding in order the following reagents:

      5% alcoholic hematoxylin -------------------- 20 ml

     10% ferric chloride ---------------------------- 8 ml

      Weigert’s iodine solution ------------------- 8 ml

      Mix the above amounts (or needed proportions thereof) well. Solution should be jet black. Use immediately and discard after use.

2% aqueous ferric chloride (prepare fresh, not necessary):

      10% ferric chloride from above ------------ 10 ml

      Distilled water ------------------------------- 50 ml

5% aqueous sodium thiosulfate:

Van Gieson’s counterstain:

      1% aqueous acid fuchsin --------------------- 5 ml

      Saturated aqueous picric acid -------------- 100 ml

For nervous tissues may be prepared as follows:

      1% aqueous acid fuchsin  ------------------- 15 ml

      Saturated aqueous picric acid ------------- 50 ml

      Distilled water ------------------------------- 50 ml

Procedure:

1.    Deparaffinize and hydrate slides to distilled water.

2.    Stain in Verhoeff’s solution for 1 hour. Tissue should be completely black.

3.    Rinse in tap water with 2-3 changes.

4.    Differentiate in 2% ferric chloride for 1-2 minutes.

5.    Stop differentiation with several changes of tap water and check microscopically for black elastic fiber staining and gray background. It is better to slightly under differentiate the tissue, since the subsequent Van Gieson’s counterstain can extract the elastic stain somewhat.

6.    Wash slides in tap water.

7.    Treat with 5% sodium thiosulfate for 1 minute. Discard solution.

8.    Wash in running tap water for 5 minutes.

9.    Counterstain in Van Gieson’s solution for 3-5 minutes.

10.  Dehydrate quickly through 95% alcohol, 2 changes of 100% alcohol.

11.  Clear in 2 changes of xylene for 3 minutes each.

12.  Coverslip with resinous mounting medium.

Results:

      Elastic fibers --------------------- blue-black to black

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

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

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

Positive Controls:

      Aorta, Kidney, Myometrium.