GFP tissue preparation for confocal microscopy
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Young Jik Kwon |
GFP tissue preparation for confocal microscopy
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All,
We are trying to take confocal micrographs of at tumor tissues that express GFP. What would be the sample preparation procedures? We tried cryosectioned samples but the cell morphology seems weird. We already know paraformaldehyde fixation kills GFP fluorescence. Any expert's advice? Best, Young |
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Cameron Nowell |
Re: GFP tissue preparation for confocal microscopy
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Hi Young, PFA fixation should not totally kill your GFP, it may weaken it
a bit but not totally wipe it out. If you find you have lost all your fluorescence
after fixation you can always go in with an anti-GFP antibody tagged with
Alexa488 or similar and get the signal back. I have fixed numerous tissues and cells in 4% PFA and seen very
little degradation of the signal. For more aggressive staining (like BrdU incorporation
that requires acid fixation) the signal is gone, this is when the anti-GFP
antibody is useful. Cheers Cam Cameron J. Nowell Office: +61 3 9341 3155 From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Young Jik Kwon All, No virus
found in this incoming message. This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd. |
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Alice Rodriguez Diaz |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by Young Jik Kwon
Dear Young,
I don't think that paraformaldehyde should be the problem. Are you using methanol? if so, methanol does kill GFP. Depending on your sample, consider switching to ethanol and related fixation protocols. Best, Alice Dr Alice Rodriguez-Diaz *Center for Biomedical Engineering * *University of Texas Medical Branch * *301 University Blvd. Rt. 1156, L17680* *Research Support Building # 21* *Galveston, Texas 77555-1156* *Center Phone: 409.772.8363 **Fax: 409.772.0751 * Young Jik Kwon wrote: > All, > > We are trying to take confocal micrographs of at tumor tissues that > express GFP. What would be the sample preparation procedures? We tried > cryosectioned samples but the cell morphology seems weird. We already > know paraformaldehyde fixation kills GFP fluorescence. Any expert's > advice? > > Best, > > Young > > |
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Young Jik Kwon |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by Cameron Nowell
Hi Cam,
Thanks for the information. We are trying to quantify gene expression by fluorescence intensity. Do you think PFA would work for such work? Best, Young
On Wed, Apr 22, 2009 at 8:01 PM, Cameron Nowell <[hidden email]> wrote:
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Cameron Nowell |
Re: GFP tissue preparation for confocal microscopy
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Hi Young, Quantifying fluorescence intensity is always difficult, if not
impossible. You will be able to measure relative shifts in intensity between
samples providing they are all treated the same, stained the same and captured
using the same settings (eg. Exposure time or gain setting etc.). PFA should be fine for all that. My recommendation is to
purchase PFA that is supplied in ampoules. We use a 16% PFA that is stored in
an ampoule under nitrogen gas. This is because PFA will oxidise to methanol
fairly quickly. This could be why you are seeing GFP loss with your fixation,
your fixative maybe all methanol by now. Cheers
Cameron J. Nowell Office: +61 3 9341 3155 From: Confocal Microscopy List
[mailto:[hidden email]] On Behalf Of Young Jik Kwon Hi Cam, On Wed, Apr 22, 2009 at 8:01 PM, Cameron Nowell <[hidden email]>
wrote: Hi Young, PFA fixation should not totally
kill your GFP, it may weaken it a bit but not totally wipe it out. If you find
you have lost all your fluorescence after fixation you can always go in with an
anti-GFP antibody tagged with Alexa488 or similar and get the signal back. I have fixed numerous tissues
and cells in 4% PFA and seen very little degradation of the signal. For more
aggressive staining (like BrdU incorporation that requires acid fixation) the
signal is gone, this is when the anti-GFP antibody is useful. Cheers Cam Cameron J. Nowell Office: +61 3 9341 3155 From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Young Jik Kwon All, No virus found in this incoming message. This communication is intended only for the named recipient
and may contain information that is confidential, legally privileged or subject
to copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any
rights if you have received this communication in error. No virus
found in this incoming message. This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waiver any rights if you have received this communication in error. The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd. |
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RICHARD BURRY |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by Young Jik Kwon
Young
Cryostat sections should appear like the live tissue. If you are doing overnight infiltration with 20% sucrose in buffer and rapid freezing with isopentane then I have no idea what the problem is. Most problems with morphology in Cryostat sections of paraformaldehyde fixed tissue comes from the freezing procedure. Dick ----- Original Message ----- From: Young Jik Kwon <[hidden email]> Date: Wednesday, April 22, 2009 10:44 pm Subject: GFP tissue preparation for confocal microscopy To: [hidden email] > All, > We are trying to take confocal micrographs of at tumor tissues that express GFP. What would be the sample preparation procedures? We tried cryosectioned samples but the cell morphology seems weird. We already know paraformaldehyde fixation kills GFP fluorescence. Any expert's advice? > Best, > Young > Spam > Not spam > Forget previous vote Richard W. Burry, Ph.D. Department of Neuroscience, College of Medicine Campus Microscopy and Imaging Facility, Director The Ohio State University Associate Editor, Journal of Histochemistry and Cytochemistry 277 Biomedical Research Tower 460 West Twelfth Avenue Columbus, Ohio 43210 Voice 614.292.2814 Cell 614.638.3345 Fax 614.247.8849 |
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In reply to this post by Young Jik Kwon
Young - We have a (yet unpublished) method for quantifying
fluorophore with a wide field, but not a confocal, microscope. You would need a
pure sample of the fluorophore (in your case, may be PFA-fixed GFP) for
calibration. But if fixation reduces its fluorescence to a variable degree it
will not work of course. Anyway, if that’s of interest to you, please
contact me directly. Mike Michael Model, Ph.D. Confocal Microscopy, Dpt Biological Sciences, 1275 University Esplanade, Kent State University, Kent, OH 44242 tel. 330-672-2874 From: Confocal Microscopy
List [mailto:[hidden email]] On Behalf Of Young Jik
Kwon Hi Cam, On Wed, Apr 22, 2009 at 8:01 PM, Cameron Nowell <[hidden email]>
wrote: Hi Young, PFA fixation should
not totally kill your GFP, it may weaken it a bit but not totally wipe it out.
If you find you have lost all your fluorescence after fixation you can always
go in with an anti-GFP antibody tagged with Alexa488 or similar and get the
signal back. I have fixed
numerous tissues and cells in 4% PFA and seen very little degradation of the
signal. For more aggressive staining (like BrdU incorporation that requires
acid fixation) the signal is gone, this is when the anti-GFP antibody is
useful. Cheers Cam Cameron J. Nowell Office: +61 3 9341 3155 From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Young Jik Kwon All, No virus found in this incoming
message. This communication is intended only for the
named recipient and may contain information that is confidential, legally
privileged or subject to copyright; the Ludwig Institute for Cancer Research
Ltd does not waiver any rights if you have received this communication in
error. |
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Stephen Bunnell |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by Young Jik Kwon
BAD PFA kills GFP fluorescence. We routinely use 1% PFA with GFP, YFP, and CFP, and have never had a problem so long as the PFA is fresh and pH’ed to between 7.0 and 7.4. -Steve On 4/22/09 10:34 PM, "Young Jik Kwon" <[hidden email]> wrote: All, **************************************************************************** Stephen C. Bunnell, Ph.D. Assistant Professor Tufts University Medical School Department of Pathology Jaharis Bldg., Room 512 150 Harrison Ave. Boston, MA 02111 Phone: (617) 636-2174 Fax: (617) 636-2990 Email: [hidden email] SHIPPING ADDRESS (for packages): Tufts University Receiving 37 Tyler St. Attn: Bunnell/Pathology/Jaharis 524 Boston, MA 02111 |
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Eric Scarfone |
Re: GFP tissue preparation for confocal microscopy
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I agree with Stephen. For all purposes, PFA should really be prepared "extemporaneously", but I find it difficult to convince student to do that these days..... Some detection problems may be linked to the amount of expression one gets. At low level the signal will be lost in the background fluorescence (green) induced by aldehydes. In this case countestaining works well. Cheers Eric > PFA does not kill GFP fluorescence. > > BAD PFA kills GFP fluorescence. > > We routinely use 1% PFA with GFP, YFP, and CFP, and have never had > a problem > so long as the PFA is fresh and pH¹ed to between 7.0 and 7.4. > > -Steve > > > > > > All, > > > > We are trying to take confocal micrographs of at tumor tissues > that express > > GFP. What would be the sample preparation procedures? We tried > cryosectioned> samples but the cell morphology seems weird. We > already know paraformaldehyde > > fixation kills GFP fluorescence. Any expert's advice? > > > > Best, > > > > Young > > > > > > > > > **************************************************************************** > Stephen C. Bunnell, Ph.D. > Assistant Professor > Tufts University Medical School > Department of Pathology > Jaharis Bldg., Room 512 > 150 Harrison Ave. > Boston, MA 02111 > > Phone: (617) 636-2174 > Fax: (617) 636-2990 > > SHIPPING ADDRESS (for packages): > Tufts University Receiving > 37 Tyler St. > Attn: Bunnell/Pathology/Jaharis 524 > Boston, MA 02111 > > > > |
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Caroline Bass |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by Stephen Bunnell
Can someone recommend a good PFA? (Product number and company please!) The ampules sound interesting... On 4/23/09 11:19 AM, "Stephen Bunnell" <Stephen.Bunnell@...> wrote: PFA does not kill GFP fluorescence. |
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Dear Caroline,
What do you mean by
dehydration precisely? Do you dry your slides after
sectioning?
Here is our ampoules
info:
(1) http://www.alfa.com, Alfa
Aesar, Item #43368
(2) http://www.emsdiasum.com,
Electron Microscopy Sciences, Item # 15710
Thanks,
Andrea
At 12:05 PM -0400 4/23/09, Caroline Bass wrote:
I concur. I regularly perfuse rats and mice with formaldehyde, and I don't have any problem getting a signal (viral vector delivered EGFP in brain, spleen, liver, muscle, etc.). I have seen severe loss with any sort of dehydration or methanol fixation in fresh tissue. Can someone recommend a good PFA? (Product number and company please!) The ampules sound interesting... -- |
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Caroline Bass |
Re: GFP tissue preparation for confocal microscopy
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I know this isn’t a very scientifically valid way of evaluating dehydration, but it seemed like a good explanation. On 4/23/09 12:15 PM, "Andrea Hooper" <anh2006@...> wrote: Dear Caroline, |
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Knecht, David |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by anh2006
I have always used the comparable Polysciences ampules of 16% formaldehyde. I don't understand the Alfa product. My understanding was that paraformaldehyde was a solid, and dissolving it led to it being converted to formaldehyde. They are selling it as 16% paraformaldehyde. Dave
On Apr 23, 2009, at 12:15 PM, Andrea Hooper wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Your understanding is correct. It's a misnomer, extremely common
and although incorrect, it's generally accepted.
At 4:04 PM -0400 4/23/09, David Knecht wrote:
I have always used the comparable Polysciences ampules of 16% formaldehyde. I don't understand the Alfa product. My understanding was that paraformaldehyde was a solid, and dissolving it led to it being converted to formaldehyde. They are selling it as 16% paraformaldehyde. Dave On Apr 23, 2009, at 12:15 PM, Andrea Hooper wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) -- |
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Sylvie Le Guyader-2 |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by Stephen Bunnell
I totally agree with Steve: only bad PFA
kills GFP. I have done a fair bit of imaging GFP in filopodia which retracts
very fast if not fixed properly. My understanding is that paraformaldehyde
is a polymer of formaldehyde and is solid. Formaldehyde is a gas that readily
dissolves in water. In a formaldehyde solution, two things
happen: - Formaldehyde equilibrates between the
air above the solution and the solution itself so every time you open your
bottle, more formaldehyde leaves the solution. Thus 15mL tubes are a good way
to store working stocks because the volume/surface ratio limits this process. - The second thing that happens is that
paraformaldehyde forms as a precipitate which is definitely a sign that your
solution needs to be trashed (not down the sink of course!). Thus I think it is
not a good idea to buy ‘4%PFA’ in big bottles as it is usually sold
except maybe if you use it very fast or if you have applications where the
quality of fixation is not critical. I prepare an 8% stock the following way: Under a fume hood, warm up 150mL of PBS to
about 60ºC Add 16g of PFA powder Stir and keep warm to dissolve Adjust the pH to 7.4 with concentrate NaOH
(you should then obtain a clear solution) Adjust the volume to 200mL with PBS Aliquote in 10 mL in 15mL falcon tubes Store the aliquots for up to several years
(currently using a 2 years old stock) at -20º The working stock can be stored at 4ºC for
a couple of months at the most I normally pre warm the amount of solution
I need to 37ºC and keep the rest in the fridge I add the warm 2x solution directly onto my
cells covered in the same volume of their own overnight medium (or changed at
least 30 min before sot that the cells are very happyJ) 5 min at 37ºC for a single layer culture
is plenty Mount and image within 24h as the autofluorescence
will quickly show up against your signal. You can image later but your signal
to noise ratio will worsen with time. I find this method excellent for
preserving filopodia. It’s also trouble free since there is no rinsing
the cells or making fresh solution every day. Good luck! Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Dept of Biosciences and Nutrition Karolinska Institutet Novum 14157 +46 (0)8 608 9240 From:
PFA does not kill GFP fluorescence. All,
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Knecht, David |
Re: GFP tissue preparation for confocal microscopy
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How long do you store the ampule formaldehyde after cracking open the vial? What temp do you store it at? Dave
On Apr 24, 2009, at 4:25 AM, Sylvie Le Guyader wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Cameron Nowell |
Re: GFP tissue preparation for confocal microscopy
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Afer cracking an ampuole i decant it into a 15/10ml tube, date it and store it in the fridge. After two weeks i throw it out.
Cheers Cam ________________________________ From: Confocal Microscopy List on behalf of David Knecht Sent: Fri 24/04/2009 10:50 PM To: [hidden email] Subject: Re: GFP tissue preparation for confocal microscopy How long do you store the ampule formaldehyde after cracking open the vial? What temp do you store it at? Dave On Apr 24, 2009, at 4:25 AM, Sylvie Le Guyader wrote: I totally agree with Steve: only bad PFA kills GFP. I have done a fair bit of imaging GFP in filopodia which retracts very fast if not fixed properly. My understanding is that paraformaldehyde is a polymer of formaldehyde and is solid. Formaldehyde is a gas that readily dissolves in water. In a formaldehyde solution, two things happen: - Formaldehyde equilibrates between the air above the solution and the solution itself so every time you open your bottle, more formaldehyde leaves the solution. Thus 15mL tubes are a good way to store working stocks because the volume/surface ratio limits this process. - The second thing that happens is that paraformaldehyde forms as a precipitate which is definitely a sign that your solution needs to be trashed (not down the sink of course!). Thus I think it is not a good idea to buy '4%PFA' in big bottles as it is usually sold except maybe if you use it very fast or if you have applications where the quality of fixation is not critical. I prepare an 8% stock the following way: Under a fume hood, warm up 150mL of PBS to about 60ºC Add 16g of PFA powder Stir and keep warm to dissolve Adjust the pH to 7.4 with concentrate NaOH (you should then obtain a clear solution) Adjust the volume to 200mL with PBS Aliquote in 10 mL in 15mL falcon tubes Store the aliquots for up to several years (currently using a 2 years old stock) at -20º The working stock can be stored at 4ºC for a couple of months at the most I normally pre warm the amount of solution I need to 37ºC and keep the rest in the fridge I add the warm 2x solution directly onto my cells covered in the same volume of their own overnight medium (or changed at least 30 min before sot that the cells are very happy:)) 5 min at 37ºC for a single layer culture is plenty Mount and image within 24h as the autofluorescence will quickly show up against your signal. You can image later but your signal to noise ratio will worsen with time. I find this method excellent for preserving filopodia. It's also trouble free since there is no rinsing the cells or making fresh solution every day. Good luck! Med vänlig hälsning / Best regards Sylvie @@@@@@@@@@@@@@@@@@@@@@@@ Sylvie Le Guyader Dept of Biosciences and Nutrition Karolinska Institutet Novum 14157 Huddinge Sweden +46 (0)8 608 9240 ________________________________ From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Stephen Bunnell Sent: 23 April 2009 17:20 To: [hidden email] Subject: Re: GFP tissue preparation for confocal microscopy PFA does not kill GFP fluorescence. BAD PFA kills GFP fluorescence. We routinely use 1% PFA with GFP, YFP, and CFP, and have never had a problem so long as the PFA is fresh and pH'ed to between 7.0 and 7.4. -Steve On 4/22/09 10:34 PM, "Young Jik Kwon" <[hidden email]> wrote: All, We are trying to take confocal micrographs of at tumor tissues that express GFP. What would be the sample preparation procedures? We tried cryosectioned samples but the cell morphology seems weird. We already know paraformaldehyde fixation kills GFP fluorescence. Any expert's advice? Best, Young **************************************************************************** Stephen C. Bunnell, Ph.D. Assistant Professor Tufts University Medical School Department of Pathology Jaharis Bldg., Room 512 150 Harrison Ave. Boston, MA 02111 Phone: (617) 636-2174 Fax: (617) 636-2990 Email: [hidden email] SHIPPING ADDRESS (for packages): Tufts University Receiving 37 Tyler St. Attn: Bunnell/Pathology/Jaharis 524 Boston, MA 02111 Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Stephen Bunnell |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by Sylvie Le Guyader-2
Her chemistry is right on, and her PFA prep is basically identical to the one we use. My only addition is- Keep the powdered PFA stock at –20°C, and don’t open the stock while the bottle is still cold. This will lead to condensation into the stock, which promotes its degradation. For preservation of fine/delicate/dynamic structures, we also use a final concentration of 4% PFA at 37°C to capture the ‘state’ instantly. We have added PFA to live cells in dynamic imaging assays, and it stops dynamic movements very quickly. If the PFA is good, you should not see _any_ fading of a GFP or YFP signal. If the PFA is good, leaving the cells in the fixative for ~24 hours does not appreciably reduce the YFP signal. Still, we usually wash it out after 10 minutes. Some antibody epitopes (empirically determined) do not fare well under these conditions- In these cases we find that a longer fix with lower doses of PFA is adequate (e.g. 0.4% PFA for 30 minutes at 37°C). I suspect this approach would fail if the troublesome epitope were it to be found in a structure such as a filopodium. -Best regards, -Steve On 4/24/09 4:25 AM, "Sylvie Le Guyader" <[hidden email]> wrote: I totally agree with Steve: only bad PFA kills GFP. I have done a fair bit of imaging GFP in filopodia which retracts very fast if not fixed properly. **************************************************************************** Stephen C. Bunnell, Ph.D. Assistant Professor Tufts University Medical School Department of Pathology Jaharis Bldg., Room 512 150 Harrison Ave. Boston, MA 02111 Phone: (617) 636-2174 Fax: (617) 636-2990 Email: [hidden email] SHIPPING ADDRESS (for packages): Tufts University Receiving 37 Tyler St. Attn: Bunnell/Pathology/Jaharis 524 Boston, MA 02111 |
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Knecht, David |
Re: GFP tissue preparation for confocal microscopy
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We usually use 1-2% formaldehyde, often with 0.1% glutaraldehyde mixed in (also from ampule aliquots) in whatever media the cells are in. This fixes slightly faster in some cells and the low glut does not generate significant background, so no quenching is necessary. It is hard to quantify any of this, but this has become our "standard" fix as it has worked well for most cell types and probes. Dave
On Apr 24, 2009, at 11:27 AM, Stephen Bunnell wrote:
Dr. David Knecht Department of Molecular and Cell Biology Co-head Flow Cytometry and Confocal Microscopy Facility U-3125 91 N. Eagleville Rd. University of Connecticut Storrs, CT 06269 860-486-2200 860-486-4331 (fax) |
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Won Yung Choi, Ph.D. |
Re: GFP tissue preparation for confocal microscopy
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In reply to this post by Young Jik Kwon
Hello, Young Jik,
I have worked for several years on endogenous CFP, YFP, RFP and GFP in transgenic mice as well as neurons (both primary cultured and in vivo) that are transfected via plasmid vectors to express YFP, GFP and dsRed and there are some important issues that I discovered that I want to share. 1) Here is how I prepare my brain sections: mice/rats are perfused with cold PBS/4% para is made fresh as everyone has pointed out. I use the brown ampules from EMS as well - but we never save them (we always use them fresh). I've found out that the brightness of the GFP is significantly reduced if the perfusion is not as good. 2) The brains are postfixed in 2% para overnight. Over fixation increases background fluorescence especially in the YFP range, but I personally have not observed overfixation reducing the signal (perhaps this is because I use fresh PFA). 3) ** I've found that using a cryostat, rather than a vibratome significantly reduces the intensity when I view the sections without immunostaining. I've done numerous testings in the lab to confirm this - I think the brains being frozen somehow affects the GFP structure, because the brains are otherwise prepared identically when I test them side by side. 4) XFP's have to be hydrated to be visible as has been elucidated by one posters. This is why a sectioned brain that is dried will not show any GFP+ cells, but once it is hydrated even with PBS, will fluoresce. Therefore, a wet mounting medium is important to use. I currently use Prolong Gold from Invritrogen - when the agent cures, it solidifies a little, but still has to be sealed with nail polish because it does not solidify completely (I am assuming this last part). I used to use Vectashield which was also good, but it remained completely wet - I have had not such experience with Fluormount G - but I think mine had been in the lab for some time so maybe a fresh one may work well. 5) Lastly, as another poster mentioned, you could use a GFP antibody to bring out the signal - however, I think it is difficult to quantify gene-expression based on the intensity of signal (with or without immunostaining) because the intensity can be lowered due to many factors mentioned above (that is, if you are working with a whole animal, and obtaining the sections - perhaps this is less of a concern in a cultured prep). As cumbersome as it may be, perhaps doing a pilot RT-PCR with GFP fluorescent cells may be enough to establish if the intensity is a reliable predictor of your gene-expression using your protocol, because however you are preparing your cells/sections may or may not be a good method. I hope this helps and good luck! Won Yung Choi ------------------------------------------------------------ Won Yung Choi, Ph.D. Research Scientist Department of Molecular Therapeutics New York State Psychiatric Institute/ Department of Psychiatry Columbia University 1051 Riverside Drive, NYSPI Unit 62 New York, NY 10032 (212) 543-6058 ------------------------------------------------------------ |
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