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Position: Discussion forums > Bioscience biotechniques discussion forums > Cell biology and histology methods forum
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Eosin in H&E stain bleeding our after coverslipping
Posted by: msadk (IP Hidden, New member, 1)
Date: July 20, 2005 12:46PM
Several months ago we acquire a 2nd Linistain Random Access Stainer for our H&E stain. We also use one for Toluidine Blue stain for Mohs frozen section histology. I cut only frozen sections at 5-6 microns. The last few weeks almost every H&E slide has almost all of the Eosin laying on top of the tissue or "bleeding out" after the slide is coversipped. I diluted Eosin-Y alcoholic with 1/2 Prepared stain and 1/2-200 proof ethanol because the stainer hold the slides in the Eosin much longer that I used to when manual staining. I did 1-2 quick dips then 3 ethanol station to clear excess stain. Now the stainer uses a conveyor belt which leaves the slides in each station for 10-15 seconds. So I diluted the stain, but it seems ever since I did that that the bleeding started or shortly thereafter. Any ideas? I used Gill 3 Hematoxylin (30 sec), 2 distilled water rinses 30 secs total, lithium bluing 15 sec, disrilled water 15 sec, 200 proof ethanol, then diluted Eosin Y followed by 3 more ethanol stations and clearing in xylene substitute solvent and xylene substitute mounting media for coverslipping. Any suggestions would be greatly appreciated. I work for a Mohs Micrographic surgeon in Albany, NY, USA. He is not bothered by the inconsistant and sometimes poor staining but I am!! Since we do not keep our frozen tissue blocks the only physical evidence of our cases besides and operative report is the slides. I fear that over time these slides will deteriorate and not store well. I would like to solve this problem just in case a legal issue ever arose for my boss or me.
Thanks. Sue Becker, B.S. HTL(ASCP) Mohs Histotechnologist c/o Dr. Michael J. Mulvaney Mohs Micrographic Surgery 2 Corporate Plaza Albany, NY 12205 USA
Re: Eosin in H&E stain bleeding our after coverslipping
Posted by: Paul Kupernik (IP Hidden, New member, 1)
Date: August 10, 2005 08:22AM
Hi Sue;
Try using a 95% rinse followed by your absolutes.
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