Re: eosin fading

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From:"Victor A. Tobias" <>
To:Barry Rittman <>
Date:Thu, 5 Aug 1999 16:49:24 -0700 (PDT)
Content-Type:TEXT/PLAIN; charset=US-ASCII

I can't totally agree on the blueing theory, because we had a problem
with eosin fading many years ago and we only use Mayer's Hematoxylin, no
blueing required. Our first thought was that we had wrong dilutions of
alcohol after the eosin. That wasn't the problem, we just made up some
fresh eosin and everything was back to normal.


On Thu, 5 Aug 1999, Barry Rittman wrote:

> I  agree with Gayle.
> The major reason that eosin does not bind well is usually that the tissue has
> retained some of the alkaline medium used for blueing the Hx.  This problem can
> be eliminated by ensuring that the eosin is slightly acidic. A few drops of 1-2%
> acetic acid in 100ml of the eosin solution usually suffices. A slight turbidity
> often results especially if using dilute eosin solutions, but this is just a
> slight precipitation of the dye base and does not interfere with staining.
> Barry
> Gayle Callis wrote:
> > One thing that is important is a good rinse after the bluing step, then going
> > into 70% rinse,or the percentage of alcohol the eosin is dissolved in
> > (sometimes not 70%) BEFORE going into the eosin y solution. Also make sure
> > the rinse after bluing solution is a minimum of 1 min, to wash away all
> > traces of the mild base used to "blue" the hematoxylin.  If one only does
> > a hand rinse instead of a running water rinse, you may want to consider
> > changing this water rinse EACH run, just to insure a correct rinse/pH
> > adjustment, and prevent carryover/incorrect pH for the eosin..
> >
> > The rationale is to have the correct pH adjustment for the
> > eosin solution.
> >
> > Learned this from Jerry Fredenburgh in a staining workshop, and afterwards
> > noticed my eosin staining was more consistent, better. One tends to not
> > think about the importance of pH when doing rinse, blue, rinse then onto
> > the counterstaining portion of H&E staining.  Comes down to some chemical
> > rules! a forte of Jerry's.
> >
> > Gayle Callis

Victor Tobias
Manager Tissue Processing Lab
Washington Animal Disease Diagnostic Lab
Washington State University
Pullman Washington 99164-7034
(509) 335-5590
(509) 335-7424 fax

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