I am posting this on behalf of Robert J. Cormier - please reply
directly to him at rcormier@audrey.lrri.org as well as the list if
you think your information is of interest to the group.
Thanks
Paul
Forwarded message:
---------------------------------------------
Hi,
I am seeking info on trypan blue staining of intact tissue sections. I
was hoping to post my questions to the cytometry list but I don't know how.
If my questions are appropriate for the list, please tell me how I can
post them. Better yet, if you know the answers or can direct me to them,
please send the info my way.
The situation is that we image Ca2+ in 400 micron brain slices by
injecting neurons with fura-2. We have had trouble lately impaling cells
with microelectrodes. We can see cells in the slices. But we don't know
about their viablility. We naively thought that if the slice was dead that
it should stain with trypan blue. Even if the dye penetrated poorly in to
the slice, at least the surface cells should stain. A 5 min application of
0.04% trypan blue did not turn the whole slice blue, not even just the
surface layer. Instead, we saw punctate staining throughout the slice,
even 100-200 microns below the surface. The vast majority of cells were
unstained. We then treated the slice with 1 mM kainate (a neurotoxin) and
restained the slice. There was an increase in in the punctate staining;
especially in areas more susceptable to kainate. All this seems to good
news for the health of our slices. But it is strange that the staining
seemed to be localized to the nuclei instead of cytoplasm. After all the
blather, the questions are: 1) Is it reasonable to use trypan blue to test
for viability in tissue slices; 2) If so, are there improvements that can
be made to our protocol; 3) If not, how can we do it; 4) Why did nuclei
stain, but not the cytoplasm?
Thanks,
Bob
----------------------------------------------------------------------
Robert J. Cormier, PhD
Lovelace Respiratory Research Institute
2425 Ridgecrest, SE
Albuquerque, NM 87108
rcormier@audrey.lrri.org
505-262-7000x5969
505-262-7043 (fax)
-----------------------------------------------------------------
J.Paul Robinson, Purdue University Cytometry Labs
Professor of Immunopharmacology
robinson@flowcyt.cyto.purdue.edu PH:765-494 6449 FAX:765-494 0517
web http://www.cyto.purdue.edu
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