Re: problem with fresh frozen eGFP sample
Posted by
Julio Vazquez on
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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
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