[Histonet] Problem with Eosin Y staining

Sherri Anderson specialstainsqueen <@t> hotmail.com
Tue Jul 19 13:03:56 CDT 2005


Eosin "bleeding" of any sort is generally caused by the presence of an 
aqueous contaminant (i.e. atmospheric moisture, inadequate dehydration after 
the eosin, etc).  So, it very well could be due to a humid environment.  
However, it would also behoove you to check the concentrations of the 
alcohols after your eosin to be sure that you have at least a couple of 
changes of 100% before the slides proceed to the clearant.  Personally, I 
always had (3) 100% alcohol positions....but since you're in a humid 
environment, you may want to increase that to four.  If you are not doing so 
already, I would also suggest covering the reagent dishes when not actively 
staining...so that the absolute alcohol does not absorb any more moisture 
from the air than it absolutely has to absorb (I would use individual dish 
covers rather than just the overall lid of the stainer...if such covers are 
available to you).  In addition, increasing the frequency with which you 
change your last series of alcohols might help (or if you "rotate", perhaps 
a total "changeout" would be in order).  Finally, I would make sure that 
your mountant is tightly capped when not in use (for the same reason as 
covering the reagent dishes when not in use -- to prevent the absorption of 
atmospheric moisture).

Most people, in their staining protocols, follow their eosin with a 95% 
alcohol.  It's the 5% water that differentiates the eosin -- increase the 
water, and you'll increase the amount of eosin that is removed from the 
tissue....decrease the water, and you'll decrease the amount of eosin that's 
removed.  The presence of water is almost certainly the culprit to the issue 
you are describing -- the challenge is finding and eliminating the source.

I hope that you find this to be somewhat helpful -- good luck!

Sherri L. Anderson, BS, HTL(ASCP)
Product Specialist
Anatomical Pathology
Thermo Electron Corp.

>From: "Habitzruther, Michael" <Michael.Habitzruther <@t> RoswellPark.org>
>To: <histonet <@t> lists.utsouthwestern.edu>
>CC: "Demant, Peter" <Peter.Demant <@t> RoswellPark.org>
>Subject: [Histonet] Problem with Eosin Y staining
>Date: Tue, 19 Jul 2005 10:56:39 -0400
>Our laboratory is having a problem with the staining of mouse tumor samples 
>using hemotoxylin and Eosin Y (H&E Stain).  Everything was going fine until 
>recently.  The specific problem we are having is after the cover-slip is 
>put on and the acrymount dries, the Eosin Y stain seems to be bleeding out, 
>leaving only the hemotoxylin staining.  A histologist from the institute 
>suggested it may have something to do with the relative humidity in our 
>lab, and therefore allowing the slides to set under a flow-hood may help.  
>Any other suggestions or protocol modifications would be greatly 
>appreciated.  Thank you very much.
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