[Histonet] Eosin too pink

[Luck, Greg D.]

	We use an 80% alcohol station immediately after the Eosin to
accomplish a similar result.  It's easy and consistent. 

-----Original Message-----
From: Monfils, Paul [mailto:PMonfils <@t> Lifespan.org] 
Sent: Friday, January 13, 2006 12:04 PM
To: 'histonet <@t> lists.utsouthwestern.edu'
Subject: RE: [Histonet] Eosin too pink

	You can try rinsing the slides in tap water after staining, as
someone already suggested.  However , the pH of tap water differs
considerably from one locality to another.  If your tap water is slightly
acidic it won't differentiate eosin at all. If it is very slightly basic (pH
7.1-7.2) you may be able to use it to remove excess eosin at a more or less
controlled rate, and will have to experiment to determine the optimum time
of exposure.  But if your tap water is more strongly basic it may remove
eosin so quickly that the differentiation is difficult to control, and
uneven differentiation is likely to occur.  I would feel safer using 95%
ethanol to partially extract eosin from overstained tissues.  Eosin
dissolves out of tissue fairly quickly in 95% ethanol, but very slowly in
100% ethanol.  In any such differentiation procedure the rack of slides
should be frequently agitated in the solvent to ensure evenness of
extraction throughout the section.

	That having been said, if I were in your position I wouldn't think
in terms of correcting the overstaining, but preventing it.  An extremely
short staining time is likely to cause uneven staining (remember, the eosin
has to extract the previous solvent, whether water or alcohol, from the
tissue before it can get into the tissue and stain it).  I would try
diluting the eosin, using whatever solvent it is made with, until the
desired intensity of staining can be achieved in a normal time period.

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