Safety equipment: Work under a hood with lab coat, gloves, and glasses.

1. De-paraffin slides in xylene (1) —————— 2 minutes (re-use)
2. De-paraffin slides in xylene (2)——————- 2 minutes (re-use)
3. Clear slides in 100% alcohol———————- 2 minutes
4. Clear slides in 100% alcohol———————- 2 minutes
5. Hydrate slides in 95% alcohol——————— 2 minutes
6. Hydrate slides in running water——————- 5 minutes
7.  VVG working solution—————————— 60 minutes (re-use)
8. Wash in running water—————————— 2 minutes
9. 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)

10. Rinse in DH2O—————————————- 1 minute

11. Sodium thiosulfate———————————– 1 minute (re-use)

12. Wash in running water—————————— 5 minutes

13. Counterstain in Van Gieson’ s——————— 1 minute (re-use)

14. Dehydrate in 95% alcohol————————– 1 minute

15.   Dehydrate in 95% alcohol———————— 1 minute

16.   Dehydrate in 100% alcohol———————- 1 minute

17.   Dehydrate in 100% alcohol———————- 2 minutes

18.   Dehydrate in 100% alcohol———————- 2 minutes

19.   Clear in xylene (3)———————————- 2 minutes

20.   Clear in xylene (4)———————————- 5 minutes

21.   Coverslip 

Results:

Elastic Fibers —————————————- Black

Background —————————————— Yellow

Notes:

1. 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.
2. Over differentiated sections can be re-stained.
3. Do not prolong the Van Gieson stain; picric acid could differentiate the stain further.
4. Be sure to follow the order of adding all reagents when making the Verhoeff’s solution.
5. To clean the Verhoeff’s stain out of the jar, wash with 2% ferric chloride solution for a few minutes.
6. For optimal results differentiate the control slide first.