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Hi everyone-
Our group has done some calcium imaging in isolated cardiac neurons from
guinea pig in the past using fluo-3 and fluo-4.  We are trying to
duplicate some of those results in atrial whole mount preparations.  I
loaded the tissue with 10 uM fluo-4 AM for 1 hour at RT in HEPES
buffered Krebs and cleaved for another hour before imaging.  What we see
is very strong fluorescence (either more dye or more Ca++) in small
cells surrounding the neurons (presumably some sort of glia) and
essentially no dye in the neurons themselves.  We applied high K to
induce action potentials in the neurons and saw no fluorescence
increases, making me believe that there's no dye in them.  It seems like
the dye is either not making it through the glial layer, or perhaps it
is being actively extruded from the neurons.  Does anyone have any ideas
about getting the dye in an keeping it in?  Next, I was going to try
loading in the refrigerator and cleaving at RT, as suggested on this
list.
Thanks in advance-
Sarah

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Sarah Locknar, Ph.D.
Director, Neuroscience COBRE Imaging / Physiology Core
College of Medicine, University of Vermont
E015 Given Building
89 Beaumont Ave.
Burlington, VT 05405
802-656-0413
802-656-8704 (fax)
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