[Histonet] Re: Decalcified Bone Eosin Overstaining

[Wait, Trevor Jordan]

Wow these are awesome, thanks everybody for feedback! So appreciated

Sent from my iPhone

> On Oct 2, 2014, at 7:05 PM, "Tony Henwood (SCHN)" <tony.henwood <@t> health.nsw.gov.au> wrote:
> 
> 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-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Wait, Trevor Jordan
> Sent: Friday, 3 October 2014 4:11 AM
> To: histonet <@t> 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 _______________________________________________
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