Re: problems with frozen mTFP1 samples
Posted by
Maria DeBernardi on
URL: http://confocal-microscopy-list.275.s1.nabble.com/problem-with-fresh-frozen-eGFP-sample-tp593098p593104.html
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Re: problem with fresh frozen eGFP sample
Hi all,
I have a user of our
Center who is interested in imaging the fluorescence distribution of an
mTFP1 (Teal Fluorescent Protein, exc max 462/emis max 492)- tagged
constructs which he injects subcutaneously into mice. Samples consist
of skin biopsies embedded in OTC medium and frozen in liquid nitrogen;
cryostat sections (~16microns) are mounted with DAPI-containing mounting medium.
Our imaging station has Hg arc lamps as light source and we can use both wide
field and confocal (Nipkow disk) mode. We can image very easily the mTFP signal
in monolayer cultures after transient transfection but have issues in
visualizing the signal in the frozen tissue. Using the same filter configuration
that works well for cells, when we go to tissue sections we have a diffuse,
unspecific fluorescence signal throughout the sample. The user claims that they
had been successful previously in having the construct (back then, tagged with
HcRed) expressed and imaged in mouse skin sections processed as described
above. I wonder if this same procedure is not compatible with mTFP imaging
conditions. Has anyone used mTFP in an analogous situation?
Thanks for your help,
Maria
Johns Hopkins University Microscopy Center Montgomery County Campus
9605
Medical Center Drive, Suite 240
Rockville, MD - 20850
[hidden email]
http://www.jhu.edu/iicmcc/
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Is the freezing destroying membrane integrity, so the
GFP leaks out of the cells? I guess it shouldn’t if fixed in formaldehyde.
If it’s the solvent, then to retain fluorescence, you could try adding a
small percentage of water to your methylbutane, if that’s possible. GFP
retains fluorescence (in our plant tissues, at least) when tissue is incubated
in up to about 85% or so ethanol, for example, but we see no fluorescence above
95% ethanol.
cheers,
Rosemary
Rosemary White
[hidden email]
CSIRO
Plant Industry
ph.
02-6246 5475
GPO Box 1600
fax.
02-6246 5334
Canberra, ACT 2601
On
11/4/08 6:03 AM, "Julio Vazquez" <[hidden email]>
wrote:
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Jean,
As far as I know, GFP fluorescence is very sensitive
to organic solvents. I suspect it is not surviving the step in Methylbutane...
You may need to find an alternative method to prepare your sample for
laser capture that doesn't involve, acids, alcohols, or other organics. I
don't have any method I can suggest, though...
--
Julio
Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA
98109-1024
http://www.fhcrc.org/
On
Apr 10, 2008, at 11:53 AM, Jean Brennan wrote:
Hi
all,
I have an animal that is Tg eGFP for a
neurotransmitter in astrocytes. It shows up very well in brain and
spinal cord after perfusing with 4%PFA. I'm trying to Laser
capture some cells for RNA work but having trouble visualizing the
cells in the fresh frozen tissue. I'm doing cervical
dislocation, the disection is under 1min., then placing the tissue into
2-Methylbuane that has been cooled and surrounded by dry ice. The
tissue is then cut in a cryostat, 8um section placed onto a slide. I
then visualize using either laser or mercury bulb (with 488 filter) and the
cells look abstract or green everywhere instead of dark background with
green astrocytes (star shaped cells). I have cut frozen sections from
PFA perfused animals several times and there is no problem with the way the
cells look. Do you think I need to hydrate the slide to
visualize the cells better? Is the freezing method
faulty? Any suggestions?
Thanks for your
help.
Jean