I increased the Acid Fuchsin step to 20 min and shorted PHO to 3 min. The results looked much better, but there still some red color of these area was washed out. How to improve this protocol? Procedure:

1. Preheat Bouin’s Fluid to 54-64°C

2. Fix slides in 2% PFA for 10 min

3. Incubate slide in preheated bouins fluid for 30 min

4. Let slide cool down for 10 min

5. Hold slide under running tap water for 2 min, until the yellow color is gone

6. Dip in MiliQ

7. Incubate slide in Hematoxilin (from kit) for 5 min

8. Hold slides under running tap water for 5 min

9. Incubate slide in Biebrich Scarlet/ Acid Fuchsin for 5 min

10. Rinse slide with MiliQ

11. Differentiate slide in phosphomolybdic/phosphotungstic acid solution for 10 min (freshly prepared)

12. Without washing, place slide in Aniline Blue for 3 min

13. Rinse slide with Mili Q

14. Apply Acetic Acid for 3 min

15. Dehydration: dip slide in 70% alcohol, 90% alcohol and 100% alcohol (dip 1min in each tube)

16. Dip slide in Xylene for 1min

17. Mount with resin