Dear Barry,

In regards to your colleague's question on using fura2 in
brain slices, while was done a long time ago, it is very
difficult to see very deeply or resolve much detail in adult
brain tissue using epifluorescence or even confocal.   With
confocal, we could only see 60 um or so into neonatal rat
brain slices, which were more translucent than adult brain
slices.

The secret to am-loading of calcium indicators has been
solved over the past decade and is now in wide use, but
virtually everyone doing this is using 2-photon imaging,
because that seems to be the best way of getting deep into
the healthiest part of brain slices.  The loading approach
was described in some early 2000's articles by Arthur
Konnerth and quite a lot of people are now using this in zebrafish
and in mouse cortex.  This is getting away from slices and more
into living animals, but the bulk-loading with the AM dyes
is now widely used, and the formula (which I lack any direct
experience with) should work just as well in vivo as in slices.
Other folks who have used this include a Kerr et al. article
on cortex input output relations, and Florian Engert in 2008
in zebrafish and Neil and Smith a few years ago in zebrafish
optic tectum.

If your user has trouble finding refs, please direct them to
me.  I will send you a recent review of mine called "Imaging
in Depth" which may be helpful (and I am happy to send it
to other list members as well-- I had promised to send a
bibliography on this to the list, but ended up just finishing
the article for a Methods in Cell Biology volume on Imaging,
Vol. 89, which has a lot of more interesting chapters than
mine!).,

Best,).,
Don

Donald M. O'Malley
Director, Behavioral Neuroscience Program
422 Richards Hall
Department of Biology
Northeastern University
617-373-2284
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www.biology.neu.edu
www.neuroscience.neu.edu