Things to ask/consider:

Does your last alcohol have any pink color in it??  If so you have water
carryover, and are contaminating your clearants.  

And do you do a gradient of alcohols after the eosin?  Richard Allan and
many other companies recommend an alcohol gradient, have 95% x 2, then
absolute X 2, to differentiate and remove eosin BEFORE the clearants. Each
company may have different variations of this, some optimized for their
stains.   

This is the way H&E staining is taught, with alcohol gradient dehydration.
You also get better contrast of the eosin if you do a gradient and much
nicer staining.  Also, don't do a few dips in each alcohol, spend a bit of
time there to remove the water and extra eosin.

 

>Date: Tue, 26 Sep 2000 13:46:42 -0400
>From: "Wylie, Teresa P" <tpw12762@glaxowellcome.com>
>Subject: eosin bleeding
>To: "'histonet@pathology.swmed.edu'" <histonet@pathology.swmed.edu>
>
>We have also had a problem with Eosin bleeding on routine H&E slides.  We
>use Xylene on the stainer and Mounting Medium from Richard-Allan on an
>automated coverslipper.  We have three absolute alcohol stations after the
>Eosin and have tried using fresh alcohols and xylenes for each staining
>rack, but still have the problem.  Sometimes it is more noticeable than
>others.  Could it be the mounting media?  I would appreciate any
>suggestions.
>
>Thanks,
>Teresa
>
> 
>Teresa P. Wylie
>Scientist
>Medicine Safety Evaluation
>GlaxoWellcome
>RTP, NC
> 
> 
>
>
>
>
>
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303