Re: problem with fresh frozen eGFP sample

Posted by Billeter-Clark Rudolf on
URL: http://confocal-microscopy-list.275.s1.nabble.com/problem-with-fresh-frozen-eGFP-sample-tp593098p593108.html

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

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

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rosemary White
Sent: 11 April 2008 00:22
To: [hidden email]
Subject: Re: problem with fresh frozen eGFP sample

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 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.     [phone]
Canberra, ACT 2601             



On 11/4/08 6:03 AM, "Julio Vazquez" <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =
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

This message has been checked for viruses but the contents of an attachment may still contain software viruses, which could damage your computer system: you are advised to perform your own checks. Email communications with the University of Nottingham may be monitored as permitted by UK legislation.