Re: problem with fresh frozen eGFP sample
Posted by
Jean Brennan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/problem-with-fresh-frozen-eGFP-sample-tp593098p593109.html
Re: problem with fresh frozen eGFP sample
Dear Rudi,
I like the idea of 2mm sections, then I can play with some different methods. I will also see if I can freeze it in the vapor of liquid nitrogen. Thank you for your help.
Best regards,
Jean
Jean Brennan
Research Specialist
Rothstein lab, Dept of Neurology
Johns Hopkins University
600 N. Wolfe St., Meyer 6-174
Baltimore, MD 21287
410-614-4119; FAX: 410-955-0672
[hidden email]>>> Billeter-Clark Rudolf <
[hidden email]> 04/11/08 5:12 AM >>>
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How about pre-slicing the tissue by hand into slices of (ideally) about 2 mm thickness and then freezing them between two flat pieces of metal (for fast cold transfer) directly in liquid nitrogen? You will get some ice cristal formation, but if I remember correctly, it is not that bad in brain tissue (I work primarily with striated muscles).
You can then lyophilise the laser capture section. This should avoid any contact with organic solvents
Rudi Billeter
School of Biomedical Sciences
University of Nottingham
Nottingham NG7 2UH
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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
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