The increased eosinophilia, in this scenario, is usually due to over-decalcification.
I would suggest: * Treat sections with 1% Periodic acid (as used in the PAS stain) for 10 minutes before Haematoxylin staining. * Use an iron-haematoxylin or Celestine blue-Haematoxylin * decrease time in eosin (or use a neutral 1% eosin stain). This should increase nuclear staining and decrease the eosin reaction. Regards Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Laboratory Manager & Senior Scientist, the Children's Hospital at Westmead Adjunct Fellow, School of Medicine, University of Western Sydney Tel: 612 9845 3306 Fax: 612 9845 3318 Pathology Department the children's hospital at westmead Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA -----Original Message----- From: histonet-boun...@lists.utsouthwestern.edu [mailto:histonet-boun...@lists.utsouthwestern.edu] On Behalf Of Wait, Trevor Jordan Sent: Friday, 3 October 2014 4:11 AM To: histonet@lists.utsouthwestern.edu Subject: [Histonet] Decalcified Bone Eosin Overstaining 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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet ********************************************************************************* This email and any files transmitted with it are confidential and intended solely for the use of the individual or entity to whom they are addressed. If you are not the intended recipient, please delete it and notify the sender. Views expressed in this message and any attachments are those of the individual sender, and are not necessarily the views of The Sydney Children's Hospitals Network. This note also confirms that this email message has been virus scanned and although no computer viruses were detected, The Sydney Childrens Hospital's Network accepts no liability for any consequential damage resulting from email containing computer viruses. ********************************************************************************* _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
