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 It has been replaced by immunohistochemisty.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.
- 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
- If sections are formalin fixed, mordant in Zenker fixative containing 5% acetic acid overnight at room temperature.
- Rinse in running water ————————— 2 minutes
- 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