This is an old stain not used much anymore for diagnosis.  It has been replaced by immunohistochemisty.

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. If sections are formalin fixed, mordant in Zenker fixative containing 5% acetic acid overnight at room temperature.
8. Rinse in running water ————————— 2 minutes
9. Place in Gram’s Iodine ————————— 15 minutes

10. Rinse in running water ————————— 1 minute

11. Place in 5% sodium thiosulfate ——————– 3 minutes

12. Wash in running water ————————— 10 minutes

13. Place in 0.25% potassium permanganate ——— 5 minutes

14. Rinse in running water ————————— 1 minute

15. Place in 5% oxalic acid ————————— 1 minute

16. Stain in PTAH at room temp ——————— Overnight

17. Dehydrate in 95% alcohol————————- 30 seconds

18. Dehydrate in 100% alcohol———————– 30 seconds

19. Dehydrate in 100% alcohol———————- 30 seconds

20. Dehydrate in 100% alcohol———————- 30 seconds

21. Clear in xylene (3)——————————- 2 minutes (re-use)

22. Clear in xylene (4)——————————- 5 minutes (re-use)

Results:

Cross-striations, fibrin – Blue

Nuclei – Blue

Collagen – Red-Brown