RE: [Histonet] Eosin Y alcoholic with or without phloxine?

From:"Monfils, Paul"

If your procedure works well on most tissues, but doesn't work on one type
of tissue from one source, I would look to the tissue, not the staining
solution, for the explanation, particularly the fixative used on the rat

The phloxine, when present, has no direct effect on the action of the eosin.
Phloxine is just used to provide a greater range of pink hues than you can
get with eosin alone.  The description of the Sigma product in the catalog
sounds pretty straightforward - 0.5% solution in acidified 90% alcohol.
That's what I use, though I mix my own solution, and I do include phloxine.

If you have a problem tissue that doesn't retain eosin well, there are a few
things you can try.  First, you can further acidify the eosin by adding 2 ml
acetic acid per 100 ml of stain. Second, after your hematoxylin bluing step
and subsequent washing, immerse the slides in 4% aqueous acetic acid for 30
to 60 seconds before going into the eosin. Third, don't leave the slides in
the dehydration alcohols longer than necessary, particularly the alcohols
less than absolute in concentration. Many labs use much longer dehydration
times than are actually necessary.  If you use 70% alcohol in your
dehydration series, skip that step. Go directly from the eosin into 95%
alcohol, and leave the slides in 95% only briefly (20 dips) before moving on
to the absolute alcohols.  95% ethanol dissolves eosin out of tissue fairly
quickly, much more quickly than absolute ethanol does.  

If the above steps don't solve the problem, you can try (1) using eosin with
phloxine.  While the phloxine doesn't affect the action of the eosin, the
tissue's inability to bind eosin doesn't necessarily mean that it won't bind
phloxine, so you may get a satisfactory stain using the combination of the
two similar dyes.  Or (2) try dehydrating with a 50:50 mix of ethanol and
isopropanol instead of pure ethanol.

> ----------
> From: on behalf of Cathy
> Malcontenti-Wilson
> Sent: 	Monday, March 27, 2006 5:34 PM
> To:
> Subject: 	[Histonet] Eosin Y alcoholic with or without phloxine?
> Dear All,
> I would like to ask about a particular problem with eosin-Y staining. I am
> using Eosin Y alcoholic without phloxine (from Sigma) and for some reason,
> all of the eosin staining comes out of the tissue as it is being 
> dehydrated. I can actually see that it just drains out. I know that this
> is 
> part of the differentiation process but it just all comes out. We use the 
> same staining set for mouse liver tumour tissues with no problem. These
> are 
> routinely processed (by a hospital pathology lab) rat rectum slides.  Is 
> the phloxine important for "keeping in the stain"? The sections are quite 
> small. Should I be using a different eosin?
> Any advice on this matter would be greatly appreciated.
> Cathy
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