Eosin Y as a fluorescent stain?

Raymond B. Hester (rhester@jaguar1.usouthal.edu)
Thu, 1 Sep 1994 08:21:35 -0500 (CDT)

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Has anyone used eosin Y as a fluorescent stain? I have been given a
couple of papers from a group in The Netherlands (M.E. Boon, et.al.) in
which they used eosin Y as a cytoplasm stain together with ethidium
bromide for DNA. Their analysis was done on a Bio-Rad MRC 600 confocal
scanner. They say that the eosin produces green fluorescence and the
ethidium bromide red with the result that they have two-color images. My
experience to date has been that eosin Y-stained cells have a yellow
color when viewed with a fluorescence microscope using FITC ex. and em.
filters. We have not done much work with our ACAS (see below) using this
stain, but the little I have done seems to indicate that when eosin Y-
stained cells are excited with the 488 line, the emitted fluorescence is
very broad - from 515 to 600, and seemingly brighter at the higher
wavelengths.

Are we doing something wrong in the staining procedure and should the
stained cytoplasm actually have emission properties similiar to the familiar
FITC green fluorescence we are all used to seeing?

Should the eosin Y be the disodium salt or the free acid?

What manufacturer of eosin Y is recommended for this purpose?

Should the cells be stained with the alcohol- or water-soluble form of
eosin Y?

We have a Meridian ACAS 570 fluorescence image analysis system and would
like to know if this dye combination (eosin Y and ethidium
bromide, or eosin Y and propidium iodide) can be excited simultaneously
with the 488 (or 514) line. We have two PMTs and can do fluorescence
compensation with the ACAS.

Thanks for any help with this.

Ray in Mo-beel

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