Verhoeff van Gieson

Classification: connective tissue stain

Mechanism of staining: ionic bonding/van de Waals forces for elastin fibers

Purpose: stain elastin

Control tissue: aorta, skin, lung

Well Stained Slide

Elastic fibers blue-black to black

Nuclei blue/grey/black

Collagen
red

Muscle orange

RBCs, cytoplasm yellow


REAGENT

REAGENT

PURPOSE

MECHANISM OF STAINING

SOURCE OF ERROR

Iron Hematoxylin (Verhoeff’s)

Primary stain for elastin

A basic staining lake which is used progressively for staining elastin and nuclei

 

Omitted: Elastin fibers will not be demonstrated.

Too short: Elastin will not be demonstrated. Differentiating time with ferric choloride will need to be shorter.

Too long: Background staining. Can remove the excess hematoxylin by leaving the slide in ferric choloride for a longer time.

Ferric choloride (2%)

Differentiator

 

Removes brown discoloration

Omitted: Overstained slide. No details will be demonstrated.

Too short: Under differentiated.

Too long: Over differentiated.

Sodium thiosulphate (5%)

Omits iodine discoloration by reacting to produce water-soluble sodium tetrathionate

Removes mercury pigments

Forms complexes at the aldehyde groups

Omitted: Van Geisson stain will be dull.

Too short: Van Geisoon stain will be dull.

Too long: No effect.

Van Geisson

Counterstain to visualize collagen, cytoplasm and muscles

(contains two acid dyes such as picric acid and acid fuchsin)

Ionic bonding  & porosity

Omitted: Tissue components will not be demonstrated.

Too long: Counterstain will obscure the primary stain and fine elastin fibers will be removed.

Too short: Background tissues will stain less intensely.

 

 

 

Special Considerations


Purpose of VVG: Demonstration of pathologic elastin fibers, atrophy of elastic fibers, arterioclerosis or other vascular disorders, breaking of elastic fibers due to ageing process and demonstration of elastic fibers in normal tissues (veins and arteries)

Proper preparation of Verhoeff’s Iron hematoxylin solution is critical for successful staining. Usually  20ml of alcoholic Hematoxylin is mixed with 8ml of Ferric chloride(10%) and 8ml of lugols Iodine. This compound should be prepared prior to use and the ingredients must be added in order because if iodine is added first, the hematoxylin immediately would be overoxidized and a substance without staining properties would be made.
The counterstain (Van Gesson) is usually used with pH between 1.0 & 2.0 which increases the number of dye binding sites for collagen. When doing the dehydration, it should be done rapidly so that the picric acid will not be lost from cytoplasm. 

There is no need to remove mercury pigments by a separate procedures because Iodine will automatically remove these pigments during the staining method.

Clean glassware and staining tray with 2% FeCl3


References

Officer B. HIML251 Lecture notes: Elastic Stains: Verhoeff’s van Gieson & Gomori’s Aldehyde Fuchsin, January 21, 2009