Donald,

This problem is more common than you might think, although not that easy to
solve.  The dead cell stains in MP's LIVE/DEAD kits are non-cell permeant,
like propidium iodide, and not set up for the kind of staining you are
doing, so probably won't give much better results.  One possible solution
is to use a mitochondrial probe, such as rhodamine 123, Mitotracker Red or
JC-1.  The advantage to using one of these dyes is that they are
membrane-permeant and may possibly be able to penetrate into more layers of
the fish.  The first two only stain healthy mitochondria;  JC-1 stains all
mitochondria, but those with high membrane potential are red and those with
low membrane potential are green. Presumabably your dead cells will have
mitochondria with low or non-existent membrane potential and will be
distinguishable from live cells.  By using JC-1, you have a control for
cell permeability.

There is even one paper using JC-1 in whole mice to study apoptosis in
spleenocytes:  N. Zamzami, et al. J Exp Med 182, 367 (1995).  The JC-1 was
injected into the mouse.

You can find more information on the mitochondrial stains at the MP web
site:
http://www.probes.com
--
Jill Hendrickson
Molecular Biology Product Manager
Molecular Probes, Inc.
Phone:  541-465-8356
Fax:    541-465-4593

Donald O'Malley wrote:

> Hi All,
>
>     We are trying to stain dead/dying cells in living zebrafish.
> We would like to do this non-invasively, i.e. by simply incubating
> the larval fish in the "dead-cell" stain.  We've tried propidium iodide
> over a range of concentrations and for times from several hours
> to overnight, all to no avail.  Presumably it can't diffuse into the
> fish, although we know that some compounds are able to diffuse
> into the fish.   I realize this is a bit esoteric, but any suggestions
> would be appreciated.   I also know there are live-dead kits from
> Molecular Probes, but before we spend hundreds of dollars, I
> thought I might check first with the imaging community.  (We are
> gently killing the cells with a low-intensity laser beam; we want
> to confirm cell death before doing extensive behavioral testing).
>
> Thanks,
> Don
>
> Donald M. O'Malley, Ph.D.
> Department of Biology
> 414 Mugar Hall
> Northeastern University
> Boston, MA 02115
> [log in to unmask]