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The page below is a sample from the LabCE course Histology Special Stains: Connective Tissue. Access the complete course and earn ASCLS P.A.C.E.-approved continuing education credits by subscribing online.

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Verhoeff-Van Gieson (VVG) Stain - Staining Protocol

Sample type required: Deparaffinized and rehydrated tissue section (3-5 microns) on positively (+) charged slides

Preferred fixative: 10% neutral buffered formalin (NBF)

Control: Aorta, artery or skin

ReagentTimeTechnical Notes
Verhoeff's elastic staining solution

1 hour
Make fresh; save solution until staining is complete.
Ferric chloride is required as a mordant in this (hematoxylin-based) solution.
Running water washUntil solution drains clear
2% ferric chloride solution
Differentiate until black fibers are crisp and well defined and background is gray

Review slides microscopically to ensure differentiation is complete.
Due to varying elastic fiber content, the control slide and patient slide should be checked individually and microscopically
to determine accurate differentiation.
If differentiation is not complete, return to solution for 30 seconds and repeat. If over-differentiation occurs, return slide(s) to Verhoff solution.

Running water washUntil solution drains clear
5% sodium thiosulfate
1 minute Removes iodine
Running water wash5 minutes
Van Gieson's solution
5 minutesCounterstain

Post staining procedure: Tissue section should be rinsed well in distilled water, dehydrated through graded alcohols(beginning with 95%). Follow with two changes of xylene and then coverslip.
    Expected results:
    • Elastic fibers and nuclei - Black
    • Collagen - Red
    • Other tissue elements - Yellow