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Safety equipment: Work under a hood with lab coat, gloves, and glasses.
- De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
- De-paraffin slides in xylene (2)——————- 2 minutes (re-use)
- Clear slides in 100% alcohol———————- 2 minutes
- Clear slides in 100% alcohol———————- 2 minutes
- Hydrate slides in 95% alcohol——————— 2 minutes
- Hydrate slides in running water——————- 5 minutes
- VVG working solution—————————— 60 minutes (re-use)
- Wash in running water—————————— 2 minutes
- Differentiate sections microscopically in 2% ferric chloride until the elastic fibers are distinct and the background is colorless to light grey. If the sections are differentiated too far, re-stain. (see notes below)
- Rinse in DH2O—————————————- 1 minute
- Sodium thiosulfate———————————– 1 minute (re-use)
- Wash in running water—————————— 5 minutes
- Counterstain in Van Gieson’ s——————— 1 minute (re-use)
- Dehydrate in 95% alcohol————————– 1 minute
- Dehydrate in 95% alcohol———————— 1 minute
- Dehydrate in 100% alcohol———————- 1 minute
- Dehydrate in 100% alcohol———————- 2 minutes
- Dehydrate in 100% alcohol———————- 2 minutes
- Clear in xylene (3)———————————- 2 minutes
- Clear in xylene (4)———————————- 5 minutes
- Coverslip
Results:
Elastic Fibers —————————————- Black
Background —————————————— Yellow
Notes:
- It is easy to over differentiate. If the background is completely colorless so that a clear yellow counterstain is obtained, the section may be over differentiated. It is probably better to err on the side of under differentiating.
- Over differentiated sections can be re-stained.
- Do not prolong the Van Gieson stain; picric acid could differentiate the stain further.
- Be sure to follow the order of adding all reagents when making the Verhoeff’s solution.
- To clean the Verhoeff’s stain out of the jar, wash with 2% ferric chloride solution for a few minutes.
- For optimal results differentiate the control slide first.
