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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) Staining - 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: Artery or skin

ReagentTimeTechnical Notes
Verhoeff (iron) hematoxylin

30 minutes
Make fresh; save solution until staining is complete.
Ferric chloride in this particular hematoxylin is required as a mordant.
Running water washUntil solution drains clear
2% ferric chloride solution
Differentiate until black fibers are well defined and background is gray

Review slides microscopically to ensure differentiation is complete.
If differentiation is not complete, return to solution for 30 seconds and repeat.

Running water washUntil solution drains clear
5% hypo (sodium thiosulfate)
1 minute Removes iodine
Running water wash1 minute
Van Gieson's solution
5 minutesCounterstain

Post staining procedure: Tissue section should be dehydrated with 95% and absolute alcohols. Follow with two changes of xylene and then coverslip.
    Expected results:
    • Elastic fibers and nuclei - Black
    • Collagen - Red
    • Other tissue elements - Yellow