Re: problems with frozen mTFP1 samples

Posted by Jean Brennan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/problem-with-fresh-frozen-eGFP-sample-tp593098p593105.html

Dear Maria,

 

Thank you for your help.  I will try your suggestion.

 

All the best,

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: [phone]
[hidden email]

>>> Maria DeBernardi <[hidden email]> 04/14/08 11:18 AM >>>

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

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

 

Maria A. DeBernardi, Ph.D.

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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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rosemary White
Sent: Thursday, April 10, 2008 7:22 PM
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