[Histonet] Decalcified Bone Eosin Overstaining

[Wait, Trevor Jordan]

Hey histonetters! I'm currently doing some H&E Staining for EDTA Decalcified Bone Tissue and it seems that the tissue is staining very intensely with Eosin...perhaps too much. Was curious to see from some of the more experienced histotechnicians out there which methods you guys have used in the past to perhaps fix an issue like this. Here is my protocol that I have used once I have sectioned the paraffin embedded tissue. I have omitted the 1 second Acid Alcohol Dip in a few of my trials and it seemed that this method worked great to keep the tissue from overstaining with Eosin. However, this is an important step for differentiation of the Hematoxylin...so I'm a little puzzled about what to do next. Perhaps wash with tap water again after the Acid Alcohol?


1. Xylene - 3 Minutes

2. Xylene 3- Minutes

3. Xylene - 3 Minutes

4. 100% EtOH - 3 Minutes

5. 100% EtOH - 3 Minutes

6. 95% EtOH - 3 Minutes

7. 70 % EtOH - 3 Minutes

8. Filtered Harris Hematoxylin (Commercial Purchase) - 6 Minutes

9. Wash with running Tap water

10. 1 second dip in 0.5% 12M HCL Acid Alcohol

11. Eosin Y Solution - 1 Minute

12. 70% EtOH - 2 Minutes

13. 70% EtOH - 2 Minutes

14. 95% EtOH - 2 Minutes

15. 95% EtOH - 2 Minutes

16. 100% EtOH - 2 Minutes

17. 100% EtOH - 2 Minutes

18. Xylene - 1 Minute

19. Xylene - 1 Minute

20. Xylene - 1 Minute


Trevor Jordan Wait
University of Texas Health Science Center, San Antonio
Class of 2017 MD Candidate
Abilene Christian University Class of 2013 Graduate
B.S.  Biochemistry