problem with fresh frozen eGFP sample
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Jean Brennan |
problem with fresh frozen eGFP sample
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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
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] |
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Julio Vazquez |
Re: problem with fresh frozen eGFP sample
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Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
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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:
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Rosemary.White |
Re: problem with fresh frozen eGFP sample
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Search the CONFOCAL archive at
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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: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = |
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Haberman, Ann |
Re: problem with fresh frozen eGFP sample
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In reply to this post by Jean Brennan
Search the CONFOCAL archive at
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Dear Jean,
We struggled with this exact issue for quite a while before we
found the solution on the internet (embedded in someone's blog about
their frustrations!).
My understanding is that acetone or 2-methybutane achieve
fixation by dehydrating tissue. As a result, that fixation process
extracts cytoplasmic GFP. For that reason, any stains for water
soluble proteins are best performed with PFA or formalin fixed tissue.
That is the case for many proteins found within the cytoplasm.
However, GFP does not fluoresce well after formaldehyde fixation
so many investigators have included an additional anti-GFP stain to
detect the presence of GFP that no longer can fluoresce! Rockland
makes an anti-GFP that also detects YFP.
best,
Ann
Content-type: text/plain; charset=utf-8
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 Jean Brennan -- Ann Haberman, PhD Department of Laboratory Medicine Yale University School of Medicine 1 Gilbert St. TAC S541 New Haven, CT 06510 203-785-7349 203-785-5415 (fax) [hidden email] |
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Billeter-Clark Rudolf |
Re: problem with fresh frozen eGFP sample
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In reply to this post by Rosemary.White
Search the CONFOCAL archive at
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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 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 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: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = 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. |
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Ian Dobbie-2 |
Re: problem with fresh frozen eGFP sample
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In reply to this post by Jean Brennan
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Jean Brennan <[hidden email]> writes: > 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, GFP fluorescence is completely dependant upon structural water within the beta barrel[1]. I'm not sure how strongly you are dehydrating your sample, but this could cause you to loose you GFP signal. Ian [1] see Tsien, RY. THE GREEN FLUORESCENT PROTEIN Annu. Rev. Biochem. 1998.67:509-544. |
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Maria DeBernardi |
Re: problems with frozen mTFP1 samples
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In reply to this post by Rosemary.White
Search the CONFOCAL archive at
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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
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 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: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = |
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Jean Brennan |
Re: problems with frozen mTFP1 samples
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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: 410-955-0672 [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
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 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: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = |
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Jean Brennan |
Re: problem with fresh frozen eGFP sample
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In reply to this post by Ian Dobbie-2
Hi Ian,
I do see that now. I will try some tissue in liquid Nitrogen and some in the vapor of liquid nitrogen to see if I can avoid water crystals. It becomes interesting when I need the fresh tissue for RNA.
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: 410-955-0672 [hidden email] >>> Ian Dobbie <[hidden email]> 04/14/08 6:40 AM >>> Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Jean Brennan <[hidden email]> writes: > 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, GFP fluorescence is completely dependant upon structural water within the beta barrel[1]. I'm not sure how strongly you are dehydrating your sample, but this could cause you to loose you GFP signal. Ian [1] see Tsien, RY. THE GREEN FLUORESCENT PROTEIN Annu. Rev. Biochem. 1998.67:509-544. |
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Jean Brennan |
Re: problem with fresh frozen eGFP sample
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In reply to this post by Billeter-Clark Rudolf
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 >>> 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 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: Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal = 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. |
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Jean Brennan |
Re: problem with fresh frozen eGFP sample
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In reply to this post by Haberman, Ann
Dear Ann,
I have tried the anti-GFP it does work, but we were trying to avoid the staining step. I have some other suggestions using liquid nitrogen and liquid nitrogen vapor. I will try that first, if that doesn't work we will use the staining method. Thank you kindly 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] >>> Ann Haberman <[hidden email]> 04/10/08 8:08 PM >>> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
Dear Jean,
We struggled with this exact issue for quite a while before we found the solution on the internet (embedded in someone's blog about their frustrations!).
My understanding is that acetone or 2-methybutane achieve fixation by dehydrating tissue. As a result, that fixation process extracts cytoplasmic GFP. For that reason, any stains for water soluble proteins are best performed with PFA or formalin fixed tissue. That is the case for many proteins found within the cytoplasm.
However, GFP does not fluoresce well after formaldehyde fixation so many investigators have included an additional anti-GFP stain to detect the presence of GFP that no longer can fluoresce! Rockland makes an anti-GFP that also detects YFP.
best,
Ann
Content-type: text/plain; charset=utf-8
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 Jean Brennan -- Ann Haberman, PhD Department of Laboratory Medicine Yale University School of Medicine 1 Gilbert St. TAC S541 New Haven, CT 06510 203-785-7349 203-785-5415 (fax) [hidden email] |
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Jean Brennan |
Re: problem with fresh frozen eGFP sample
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In reply to this post by Rosemary.White
Hi Rosemary,
I did try the alochols and they still don't work. I'll try some instant freeze methods with Liquid nitrogen and hope for little water crystals. I think I will also try liquid nitrogen vapor to try to avoid the crystilization too. 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] >>> Rosemary White <[hidden email]> 04/10/08 7:21 PM >>> 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. 02-6246 5334 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 = |
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Tim O'Brien Sr. |
Carolina Workshop on Force Measurements and Manipulation in Biological Microscopy
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In reply to this post by Jean Brennan
Search the CONFOCAL archive at
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Just an update on our workshop. We have a few prime seats available for
our workshop, and a keynote speaker to announce. John Weisel has been
a leader in the fibrin world for many years, and has the leading model
for how the protein assembles from monomers to clots. Please tell
anyone interested in probing the role of forces on single molecules,
complex fluids, or cells!
Thanks, Tim O'Brien UNC Department of Physics and Astronomy Carolina Workshop on Force Measurements and Manipulation in Biological Microscopy at UNC Chapel Hill on May 27-30, 2008. The workshop is hosted by our NIH resource CISMM (Computer Integrated Systems for Microscopy and Manipulation) See http://www.cs.unc.edu/Research/nano/cismm/index.html for an overview of CISMM. The workshop consists of morning lectures that provide a framework for understanding and analyzing forces on the micro and nanoscale, and serve as an introduction to the afternoon’s experiments. The afternoon sessions are hands-on laboratories where you work with live samples: single molecules of DNA, epithelial cells, and biological fibers such as fibrin fibers on structured surfaces. Experiments use laser tweezers, an integrated atomic force microscopy/optical microscopy system, and 3D magnetic systems integrated with a fluorescence confocal microscope. We also include an introduction to microfluidics. Finally, participants spend a half day learning to use some of the many free software packages available from our resource that facilitate the analysis of forces in biological systems. Registration is limited to 18, so you have lots of hands-on time with the microscopes. The $775 fee includes light breakfasts, snacks and one dinner. Our keynote speaker will be John Weisel of the Dept. of Cell & Developmental Biology at the University of Pennsylvania school of Medicine. http://www.cs.unc.edu/Research/nano/cismm/ForcesWorkshop.htm . Hope to see you there! |
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heckman@bgnet.bgsu.edu |
Re: problem with fresh frozen eGFP sample
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In reply to this post by Haberman, Ann
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
BTW, GFP does fluoresce after PFA, as long as the formaldehyde is
made up from paraformaldehyde and the fixation time is restricted to
10 minutes.
Carol
Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear Jean, We struggled with this exact issue for quite a while before we found the solution on the internet (embedded in someone's blog about their frustrations!). My understanding is that acetone or 2-methybutane achieve fixation by dehydrating tissue. As a result, that fixation process extracts cytoplasmic GFP. For that reason, any stains for water soluble proteins are best performed with PFA or formalin fixed tissue. That is the case for many proteins found within the cytoplasm. However, GFP does not fluoresce well after formaldehyde fixation so many investigators have included an additional anti-GFP stain to detect the presence of GFP that no longer can fluoresce! Rockland makes an anti-GFP that also detects YFP. best, Ann
--
-- Carol A. Heckman, Ph.D.
Director, Center for Microscopy & Microanalysis and Professor of Biological Sciences Bowling Green State University Bowling Green, OH 43403 USA website: http://www.bgsu.edu/departments/biology/facilities/MnM |
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