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Verhoeff Van Gieson Elastic Stain

Verhoeff van gieson elastic stain post we have briefly explained about Verhoeff van gieson stain method’s principle, requirements, verhoeff-van gieson elastic stain procedure, result with label.

Verhoeff Van Gieson Elastic Stain

Verhoeff-van gieson elastic stain is useful in demonstrating elastic tissue atrophy in cases of emphysema, as well as the thinning and loss of elastic fibres in arteriosclerosis and other vascular diseases. Changes such as splitting and fragmentation occur as we age; these changes are most visible in the skin, which becomes wrinkled and ‘loose-fitting.

Principle

The tissue is stained with a regressive hematoxylin, consisting of ferric chloride and iodine. The differentiating is accomplished by using excess mordant (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 of the iron hematoxylin complex and will retain the dye longer than the other tissue elements.

Reagents

5% Alcoholic hematoxylin

Hematoxylin: 5gm

100% alcohol: 100 ml

5% Alcoholic hematoxylin:  Dissolve 5gm of hematoxylin into 100 ml of 100% alcohol with the aide of gentle heat, do not boil. Label with date and initials, solution are stable for 1 year. (CAUTION: Flammable, avoid contact and inhalation).

10% Ferric Chloride

Ferric chloride: 10.0 gm

Distilled water: 100 ml

Dissolve 10.0 gm Ferric chloride with 100 ml of double distilled water.  Label with date and initials, solution are stable for 1 year. CAUTION: Avoid contact and inhalation.

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

verhoeff-van gieson elastic stain 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

2% aqueous ferric chloride: 10 ml of 10% ferric chloride from above mixed well with 50 ml Distilled water.

5% aqueous sodium thiosulfate

Dissolve 24.8g of sodium thiosulphate pentahydrate in 800 ml of freshly boiled and cooled water and mix thoroughly by shaking for approximately 15 minutes.

Van Gieson's counterstain

1% Acid fuchsin: 1.0 ml

Picric acid, saturated: 45.0 ml

1.0 ml 1% Acid fuchsin mixed well with 45.0 ml Picric acid, saturated, Allow to stand overnight. Label with date and initials. This verhoeff-van gieson elastic stain counterstain is Stable for 2 weeks.

Procedure

1. Deparaffinize and hydrate slides to distilled water. Stain in verhoeff-van gieson elastic stain for 1 hour. Tissue should be completely black.

2. Rinse in tap water with 2-3 changes and Rinse in distilled water with 2-3 changes. Differentiate in 2% ferric chloride for 1-2 minutes.

3. Stop differentiation with several changes of tap water and check microscopically for black elastic fiber staining and gray background.

4. It is better to slightly under differentiate the tissue, since the subsequent Van Gieson’s counterstain can extract the elastic stain somewhat.

5. Wash slides in tap water. Treat with 5% sodium thiosulfate for 1 minute. Discard solution. Wash in running tap water for 5 minutes.

6. Counterstain in Van Gieson’s solution for 3-5 minutes. Dehydrate quickly through 95% alcohol, 2 changes of 100% alcohol.

7. Clear in 2 changes of xylene for 3 minutes each. Coverslip with permanent mounting medium (DPX).

Result

Verhoeff-Van Gieson (VVG) Staining

Elastic fibers: Blue-black to black

Nuclei: Blue to black

Collagen: Red

Other tissue elements: Yellow

Further Readings

Reference