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GFP/RFP quenching

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Scott Howell-3

GFP/RFP quenching

List,

We have had some issues here with a lab where the GFP/RFP brightness in
fixed cells has become extremely variable. Like night and day. Wondering
if it may be related to the pH of their paraformaldehyde fix? Does one
particular pH seem to work best?? Any other ideas? Thanks.

Scott J. Howell, Ph.D.
Manager, Imaging Module
Visual Sciences Research Center
Case Western Reserve University
2085 Adelbert Rd.
Institute of Pathology Room 106
Cleveland, Ohio 44106
216-368-2300
http://www.case.edu/med/vsrc/

Kelly Lundsten

Re: GFP/RFP quenching

Hi Scott,

Any denaturant will be a potential problem for the protein. This
includes any MeOH that might be used to stabilize the PFA. Have them
make up fresh PFA the hard way, with heat and time, or buy it with a no
MeOH used guarantee. Also, if they are sealing their coverslips with
nail polish, stop that. Use VALAP (Vaseline:lanolin:paraffin) or
something else inert. The nail polish can seep under the coverslip over
time, denaturing the GFP. This sounds like the more likely culprit in
this instance if there is signal right after coverslipping but that
signal fades quickly over time. Or, use an antifade that cures, one
that doesn't require sealant. I believe Prolong Gold fits that criteria
(cures without dehydration around the edges of the coverslip). If the
problem persists after trying these potential solutions, then the PFA
might be changing the efficiency of the GFP due to its cross-linking
effect. Trying lower percent concentration of the PFA would be your
next step.

Good luck,
Kelly Lundsten

Sr. App Scientist
ART, Inc.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Scott Howell
Sent: Wednesday, March 04, 2009 6:52 AM
To: [hidden email]
Subject: GFP/RFP quenching

List,

We have had some issues here with a lab where the GFP/RFP brightness in
fixed cells has become extremely variable. Like night and day. Wondering
if it may be related to the pH of their paraformaldehyde fix? Does one
particular pH seem to work best?? Any other ideas? Thanks.

Scott J. Howell, Ph.D.
Manager, Imaging Module
Visual Sciences Research Center
Case Western Reserve University
2085 Adelbert Rd.
Institute of Pathology Room 106
Cleveland, Ohio 44106
216-368-2300
http://www.case.edu/med/vsrc/

dmart05

Re: GFP/RFP quenching

In reply to this post by Scott Howell-3

Scott,

Regular paraformaldehyde will quench the GFP signal because of the
methanol that forms in it. We use methanol free formaldehyde, 10%,
ultrapure from Polysciences.

Any alcohol will quench the GFP signal.

Since this formaldehyde comes already in solution, it also saves you
the time and trouble of preparing the paraformaldehyde yourself. We
just dilute it to 4% in PBS pH 7.4 with no calcium or magnesium, and
use it immediately. I always make up only what I need, and don't try to
store the diluted formaldehyde.

Deb Martinson
Research Specialist
Internal Medicine Imaging Core
Division of Cardiology
Department of Medicine
Emory University School of Medicine
101 Woodruff Circle WMB 303
Atlanta, GA 30322

404-727-3712

Quoting Scott Howell <[hidden email]>:

> List,
>
> We have had some issues here with a lab where the GFP/RFP brightness in
> fixed cells has become extremely variable. Like night and day. Wondering
> if it may be related to the pH of their paraformaldehyde fix? Does one
> particular pH seem to work best?? Any other ideas? Thanks.
>
> Scott J. Howell, Ph.D.
> Manager, Imaging Module
> Visual Sciences Research Center
> Case Western Reserve University
> 2085 Adelbert Rd.
> Institute of Pathology Room 106
> Cleveland, Ohio 44106
> 216-368-2300
> http://www.case.edu/med/vsrc/
>
>
>

Phillips, Thomas E.

Re: GFP/RFP quenching

Paraformaldehyde has no methanol. Formalin does and therefore it is
incorrect to use the terms interchangeably.

Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Chair, MU Faculty Council
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of [hidden email]
Sent: Wednesday, March 04, 2009 9:43 AM
To: [hidden email]
Subject: Re: GFP/RFP quenching

Scott,

Regular paraformaldehyde will quench the GFP signal because of the
methanol that forms in it. We use methanol free formaldehyde, 10%,
ultrapure from Polysciences.

Any alcohol will quench the GFP signal.

Since this formaldehyde comes already in solution, it also saves you
the time and trouble of preparing the paraformaldehyde yourself. We
just dilute it to 4% in PBS pH 7.4 with no calcium or magnesium, and
use it immediately. I always make up only what I need, and don't try to
store the diluted formaldehyde.

Deb Martinson
Research Specialist
Internal Medicine Imaging Core
Division of Cardiology
Department of Medicine
Emory University School of Medicine
101 Woodruff Circle WMB 303
Atlanta, GA 30322

404-727-3712

Quoting Scott Howell <[hidden email]>:

> List,
>
> We have had some issues here with a lab where the GFP/RFP brightness
in
> fixed cells has become extremely variable. Like night and day.
Wondering

> if it may be related to the pH of their paraformaldehyde fix? Does one
> particular pH seem to work best?? Any other ideas? Thanks.
>
> Scott J. Howell, Ph.D.
> Manager, Imaging Module
> Visual Sciences Research Center
> Case Western Reserve University
> 2085 Adelbert Rd.
> Institute of Pathology Room 106
> Cleveland, Ohio 44106
> 216-368-2300
> http://www.case.edu/med/vsrc/
>
>
>

Carol Heckman

Re: GFP/RFP quenching

In reply to this post by Kelly Lundsten

Hi, Scott-
We have found that limiting the duration of formaldehyde fixation to 10 minutes does conserve the GFP signal. It seems to decline the longer the cells sit in the fixative.
Carol Heckman
Center for Microscopy & Microanalysis
Bowling Green State University
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Kelly Lundsten [[hidden email]]
Sent: Wednesday, March 04, 2009 10:11 AM
To: [hidden email]
Subject: Re: GFP/RFP quenching

Hi Scott,

Any denaturant will be a potential problem for the protein. This
includes any MeOH that might be used to stabilize the PFA. Have them
make up fresh PFA the hard way, with heat and time, or buy it with a no
MeOH used guarantee. Also, if they are sealing their coverslips with
nail polish, stop that. Use VALAP (Vaseline:lanolin:paraffin) or
something else inert. The nail polish can seep under the coverslip over
time, denaturing the GFP. This sounds like the more likely culprit in
this instance if there is signal right after coverslipping but that
signal fades quickly over time. Or, use an antifade that cures, one
that doesn't require sealant. I believe Prolong Gold fits that criteria
(cures without dehydration around the edges of the coverslip). If the
problem persists after trying these potential solutions, then the PFA
might be changing the efficiency of the GFP due to its cross-linking
effect. Trying lower percent concentration of the PFA would be your
next step.

Good luck,
Kelly Lundsten

Sr. App Scientist
ART, Inc.

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]]
On Behalf Of Scott Howell
Sent: Wednesday, March 04, 2009 6:52 AM
To: [hidden email]
Subject: GFP/RFP quenching

List,

We have had some issues here with a lab where the GFP/RFP brightness in
fixed cells has become extremely variable. Like night and day. Wondering
if it may be related to the pH of their paraformaldehyde fix? Does one
particular pH seem to work best?? Any other ideas? Thanks.

Scott J. Howell, Ph.D.
Manager, Imaging Module
Visual Sciences Research Center
Case Western Reserve University
2085 Adelbert Rd.
Institute of Pathology Room 106
Cleveland, Ohio 44106
216-368-2300
http://www.case.edu/med/vsrc/

Adrian Smith-6

Re: GFP/RFP quenching

In reply to this post by Phillips, Thomas E.

On 05/03/2009, at 3:27 AM, Phillips, Thomas E. wrote:

> Paraformaldehyde has no methanol. Formalin does and therefore it is
> incorrect to use the terms interchangeably.
>
> Thomas E. Phillips, Ph.D

Good point.

<http://www.ncbi.nlm.nih.gov/pubmed/12769269?dopt=abstract>

Regards,

Adrian Smith
Centenary Institute, Sydney, Australia

Steffen Dietzel

Re: GFP/RFP quenching

In reply to this post by Carol Heckman

Hello List,

concerning the nail polish: Having done
experiments on both sides of the Atlantic, I
found that the brands I used in the US would kill
the GFP-Fluorescence while the brands in Germany
did not. Must be some specific component that is
not included in all fomulas. We had this
discussion on the list before, at least once,
like 10 years ago :-) In the US, I alternatively
used candle wax, cut in stripes, put at the edge
of the coverslip and heated with a glowing paper
clip. Or, a hardening mounting medium (ProLong, MolProbes).

Concerning GFP and formaldehyde: I sometimes let
the fixation sit over night or over the week-end
without problems. This was the freshly-made-from-paraformaldehyde type.

And while we are on nomenclature, a bit more wise
cracking: there is no such thing as a
paraformaldehyde solution. Paraformaldehyde is a
polymer (white powder) that is unsoluble. Heat
transforms it to the gas formaldehyde which can be solved in water.

Cheers

Steffen

At 18:21 04.03.2009, you wrote:

>Hi, Scott-
>We have found that limiting the duration of
>formaldehyde fixation to 10 minutes does
>conserve the GFP signal. It seems to decline
>the longer the cells sit in the fixative.
>Carol Heckman
>Center for Microscopy & Microanalysis
>Bowling Green State University
>________________________________________
>From: Confocal Microscopy List
>[[hidden email]] On Behalf Of
>Kelly Lundsten [[hidden email]]
>Sent: Wednesday, March 04, 2009 10:11 AM
>To: [hidden email]
>Subject: Re: GFP/RFP quenching
>
>Hi Scott,
>
>Any denaturant will be a potential problem for the protein. This
>includes any MeOH that might be used to stabilize the PFA. Have them
>make up fresh PFA the hard way, with heat and time, or buy it with a no
>MeOH used guarantee. Also, if they are sealing their coverslips with
>nail polish, stop that. Use VALAP (Vaseline:lanolin:paraffin) or
>something else inert. The nail polish can seep under the coverslip over
>time, denaturing the GFP. This sounds like the more likely culprit in
>this instance if there is signal right after coverslipping but that
>signal fades quickly over time. Or, use an antifade that cures, one
>that doesn't require sealant. I believe Prolong Gold fits that criteria
>(cures without dehydration around the edges of the coverslip). If the
>problem persists after trying these potential solutions, then the PFA
>might be changing the efficiency of the GFP due to its cross-linking
>effect. Trying lower percent concentration of the PFA would be your
>next step.
>
>Good luck,
>Kelly Lundsten
>
>Sr. App Scientist
>ART, Inc.
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Scott Howell
>Sent: Wednesday, March 04, 2009 6:52 AM
>To: [hidden email]
>Subject: GFP/RFP quenching
>
>List,
>
>We have had some issues here with a lab where the GFP/RFP brightness in
>fixed cells has become extremely variable. Like night and day. Wondering
>if it may be related to the pH of their paraformaldehyde fix? Does one
>particular pH seem to work best?? Any other ideas? Thanks.
>
>Scott J. Howell, Ph.D.
>Manager, Imaging Module
>Visual Sciences Research Center
>Case Western Reserve University
>2085 Adelbert Rd.
>Institute of Pathology Room 106
>Cleveland, Ohio 44106
>216-368-2300
>http://www.case.edu/med/vsrc/

--
---------------------------------------------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room (for letters etc.):
Marchioninistr. 15, D-81377 München

Building location and address for courier, parcel services etc:
Marchioninistr. 27, D-81377 München (Großhadern)

anh2006

Re: GFP/RFP quenching

I believe isopropanol is the offending ingredient in nail polish. Now it just isn't worth the risk, I never go near the stuff.

-----Original Message-----
From: Steffen Dietzel <[hidden email]>

Date: Thu, 05 Mar 2009 10:57:03
To: <[hidden email]>
Subject: Re: GFP/RFP quenching

Hello List,

concerning the nail polish: Having done
experiments on both sides of the Atlantic, I
found that the brands I used in the US would kill
the GFP-Fluorescence while the brands in Germany
did not. Must be some specific component that is
not included in all fomulas. We had this
discussion on the list before, at least once,
like 10 years ago :-) In the US, I alternatively
used candle wax, cut in stripes, put at the edge
of the coverslip and heated with a glowing paper
clip. Or, a hardening mounting medium (ProLong, MolProbes).

Concerning GFP and formaldehyde: I sometimes let
the fixation sit over night or over the week-end
without problems. This was the freshly-made-from-paraformaldehyde type.

And while we are on nomenclature, a bit more wise
cracking: there is no such thing as a
paraformaldehyde solution. Paraformaldehyde is a
polymer (white powder) that is unsoluble. Heat
transforms it to the gas formaldehyde which can be solved in water.

Cheers

Steffen

At 18:21 04.03.2009, you wrote:

RICHARD BURRY

Re: GFP/RFP quenching

In reply to this post by Steffen Dietzel

Listers

I must jump in with an different view. Comments about the terms for formaldehyde fixation have been theoretical. However, when you see what people are doing in a core facility daily, the practical becomes more important! It would be nice if people doing immunocytochemistry or fixing GFP knew the difference between formalin and paraformaldehyde as a source of formaldehyde. However, users learning microscopy need different terms to describe the sources of formaldehyde. I recommend that rather than using formaldehyde, the terms formalin and paraformaldehyde be used to describe the fixative in articles. While this is not correct, the amount of time and money saved by people using the wrong chemical will justify being inaccurate.

Dick

----- Original Message -----
From: Steffen Dietzel <[hidden email]>
Date: Thursday, March 5, 2009 4:57 am
Subject: Re: GFP/RFP quenching
To: [hidden email]

> Hello List,
>
> concerning the nail polish: Having done
> experiments on both sides of the Atlantic, I
> found that the brands I used in the US would kill
> the GFP-Fluorescence while the brands in Germany
> did not. Must be some specific component that is
> not included in all fomulas. We had this
> discussion on the list before, at least once,
> like 10 years ago :-) In the US, I alternatively
> used candle wax, cut in stripes, put at the edge
> of the coverslip and heated with a glowing paper
> clip. Or, a hardening mounting medium (ProLong, MolProbes).
>
> Concerning GFP and formaldehyde: I sometimes let
> the fixation sit over night or over the week-end
> without problems. This was the freshly-made-from-
> paraformaldehyde type.
>
> And while we are on nomenclature, a bit more wise
> cracking: there is no such thing as a
> paraformaldehyde solution. Paraformaldehyde is a
> polymer (white powder) that is unsoluble. Heat
> transforms it to the gas formaldehyde which can be solved in water.
>
> Cheers
>
> Steffen
>
> At 18:21 04.03.2009, you wrote:
> >Hi, Scott-
> >We have found that limiting the duration of
> >formaldehyde fixation to 10 minutes does
> >conserve the GFP signal. It seems to decline
> >the longer the cells sit in the fixative.
> >Carol Heckman
> >Center for Microscopy & Microanalysis
> >Bowling Green State University
> >________________________________________
> >From: Confocal Microscopy List
> >[[hidden email]] On Behalf Of
> >Kelly Lundsten [[hidden email]]
> >Sent: Wednesday, March 04, 2009 10:11 AM
> >To: [hidden email]
> >Subject: Re: GFP/RFP quenching
> >
> >Hi Scott,
> >
> >Any denaturant will be a potential problem for the
> protein. This
> >includes any MeOH that might be used to stabilize the
> PFA. Have them
> >make up fresh PFA the hard way, with heat and time, or buy it
> with a no
> >MeOH used guarantee. Also, if they are sealing their
> coverslips with
> >nail polish, stop that. Use VALAP
> (Vaseline:lanolin:paraffin) or
> >something else inert. The nail polish can seep under the
> coverslip over
> >time, denaturing the GFP. This sounds like the more
> likely culprit in
> >this instance if there is signal right after coverslipping but that
> >signal fades quickly over time. Or, use an antifade that
> cures, one
> >that doesn't require sealant. I believe Prolong Gold fits
> that criteria
> >(cures without dehydration around the edges of the
> coverslip). If the
> >problem persists after trying these potential solutions, then
> the PFA
> >might be changing the efficiency of the GFP due to its cross-linking
> >effect. Trying lower percent concentration of the PFA
> would be your
> >next step.
> >
> >Good luck,
> >Kelly Lundsten
> >
> >Sr. App Scientist
> >ART, Inc.
> >
> >-----Original Message-----
> >From: Confocal Microscopy List
> [mailto:[hidden email]]>On Behalf Of Scott Howell
> >Sent: Wednesday, March 04, 2009 6:52 AM
> >To: [hidden email]
> >Subject: GFP/RFP quenching
> >
> >List,
> >
> >We have had some issues here with a lab where the GFP/RFP
> brightness in
> >fixed cells has become extremely variable. Like night and day.
> Wondering>if it may be related to the pH of their
> paraformaldehyde fix? Does one
> >particular pH seem to work best?? Any other ideas? Thanks.
> >
> >Scott J. Howell, Ph.D.
> >Manager, Imaging Module
> >Visual Sciences Research Center
> >Case Western Reserve University
> >2085 Adelbert Rd.
> >Institute of Pathology Room 106
> >Cleveland, Ohio 44106
> >216-368-2300
> >http://www.case.edu/med/vsrc/
>
> --
> -----------------------------------------------------------------
> ----------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Mail room (for letters etc.):
> Marchioninistr. 15, D-81377 München
>
> Building location and address for courier, parcel services etc:
> Marchioninistr. 27, D-81377 München (Großhadern)
>
>
> --
> BEGIN-ANTISPAM-VOTING-LINKS
> ------------------------------------------------------
>
> Teach CanIt if this mail (ID 817899366) is spam:
> Spam:
> https://antispam.osu.edu/b.php?c=s&i=817899366&m=0b7349967249Not
> spam: https://antispam.osu.edu/b.php?c=n&i=817899366&m=0b7349967249
> Forget vote:
> https://antispam.osu.edu/b.php?c=f&i=817899366&m=0b7349967249----
> --------------------------------------------------
> END-ANTISPAM-VOTING-LINKS
>

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

Phillips, Thomas E.

Re: GFP/RFP quenching

Dick and Steffen - If you really want to get to the fundamentals, one needs to realize that soon after making “formaldehyde” from freshly depolymerized paraformaldehyde, it become hydrated into methylene glycol that forms an equilibrium with free formaldehyde (for references, see Fox et al., 1985 J. Histochem. Cytochem. 33:845-853). My understanding is that the bulk of the molecules are not free formaldehyde. Referring to a “2% formaldehyde” solution is thus not strictly true although most of us would understand what is meant by this. The best nomenclature would be to refer to a solution of “2% freshly depolymerized paraformaldehyde” assuming one started from the crystals/granules/powder formulation. Tom

Thomas E. Phillips, Ph.D

Professor of Biological Sciences

Chair, MU Faculty Council

Director, Molecular Cytology Core

2 Tucker Hall

University of Missouri

Columbia, MO 65211-7400

573-882-4712 (office)

573-882-0123 (fax)

[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of RICHARD BURRY
Sent: Thursday, March 05, 2009 9:33 AM
To: [hidden email]
Subject: Re: GFP/RFP quenching

Listers

Dick

----- Original Message -----
From: Steffen Dietzel <[hidden email]>
Date: Thursday, March 5, 2009 4:57 am
Subject: Re: GFP/RFP quenching
To: [hidden email]

>
> And while we are on nomenclature, a bit more wise cracking: there is no such thing as a
> paraformaldehyde solution. Paraformaldehyde is a polymer (white powder) that is unsoluble. Heat
> transforms it to the gas formaldehyde which can be solved in water.
>
> Cheers
>
> Steffen
>
> At 18:21 04.03.2009, you wrote:
> >Hi, Scott-
> >We have found that limiting the duration of
> >formaldehyde fixation to 10 minutes does
> >conserve the GFP signal. It seems to decline
> >the longer the cells sit in the fixative.
> >Carol Heckman
> >Center for Microscopy & Microanalysis
> >Bowling Green State University
> >________________________________________
> >From: Confocal Microscopy List
> >[[hidden email]] On Behalf Of
> >Kelly Lundsten [[hidden email]]
> >Sent: Wednesday, March 04, 2009 10:11 AM
> >To: [hidden email]
> >Subject: Re: GFP/RFP quenching
> >
> >Hi Scott,
> >
> >Any denaturant will be a potential problem for the
> protein. This
> >includes any MeOH that might be used to stabilize the
> PFA. Have them
> >make up fresh PFA the hard way, with heat and time, or buy it
> with a no
> >MeOH used guarantee. Also, if they are sealing their
> coverslips with
> >nail polish, stop that. Use VALAP
> (Vaseline:lanolin:paraffin) or
> >something else inert. The nail polish can seep under the
> coverslip over
> >time, denaturing the GFP. This sounds like the more
> likely culprit in
> >this instance if there is signal right after coverslipping but that
> >signal fades quickly over time. Or, use an antifade that
> cures, one
> >that doesn't require sealant. I believe Prolong Gold fits
> that criteria
> >(cures without dehydration around the edges of the
> coverslip). If the
> >problem persists after trying these potential solutions, then
> the PFA
> >might be changing the efficiency of the GFP due to its cross-linking
> >effect. Trying lower percent concentration of the PFA
> would be your
> >next step.
> >
> >Good luck,
> >Kelly Lundsten
> >
> >Sr. App Scientist
> >ART, Inc.
> >
> >-----Original Message-----
> >From: Confocal Microscopy List
> [mailto:[hidden email]]>On Behalf Of Scott Howell
> >Sent: Wednesday, March 04, 2009 6:52 AM
> >To: [hidden email]
> >Subject: GFP/RFP quenching
> >
> >List,
> >
> >We have had some issues here with a lab where the GFP/RFP
> brightness in
> >fixed cells has become extremely variable. Like night and day.
> Wondering>if it may be related to the pH of their
> paraformaldehyde fix? Does one
> >particular pH seem to work best?? Any other ideas? Thanks.
> >
> >Scott J. Howell, Ph.D.
> >Manager, Imaging Module
> >Visual Sciences Research Center
> >Case Western Reserve University
> >2085 Adelbert Rd.
> >Institute of Pathology Room 106
> >Cleveland, Ohio 44106
> >216-368-2300
> >http://www.case.edu/med/vsrc/
>
> --
> -----------------------------------------------------------------
> ----------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Mail room (for letters etc.):
> Marchioninistr. 15, D-81377 München
>
> Building location and address for courier, parcel services etc:
> Marchioninistr. 27, D-81377 München (Großhadern)
>
>
> --
> BEGIN-ANTISPAM-VOTING-LINKS
> ------------------------------------------------------
>
> Teach CanIt if this mail (ID 817899366) is spam:
> Spam:
> https://antispam.osu.edu/b.php?c=s&i=817899366&m=0b7349967249Not
> spam: https://antispam.osu.edu/b.php?c=n&i=817899366&m=0b7349967249
> Forget vote:
> https://antispam.osu.edu/b.php?c=f&i=817899366&m=0b7349967249----
> --------------------------------------------------
> END-ANTISPAM-VOTING-LINKS
>

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

Howard Berg

Re: GFP/RFP quenching

The Fox paper is an excellent review and a great one to bring up, Tom. The skewed equilibrium is a good explanation for the poor crosslinking ability of formaldehyde (too few molecules in solution) and its propensity for vesiculating membranes in TEM preps (the effect of methylene glycol).

R. Howard Berg, Ph.D.

Director, Integrated Microscopy Facility

Danforth Plant Science Center

975 N. Warson Rd.

St. Louis, MO 63132

ph 314-587-1261 fx 314-587-1361 cell 314-378-2409

[hidden email] www.danforthcenter.org

visit this educational resource: http://www.danforthcenter.org/Cells/

On Mar 5, 2009, at 9:56 AM, Phillips, Thomas E. wrote:

Dick and Steffen - If you really want to get to the fundamentals, one needs to realize that soon after making “formaldehyde” from freshly depolymerized paraformaldehyde, it become hydrated into methylene glycol that forms an equilibrium with free formaldehyde (for references, see Fox et al., 1985 J. Histochem. Cytochem. 33:845-853). My understanding is that the bulk of the molecules are not free formaldehyde. Referring to a “2% formaldehyde” solution is thus not strictly true although most of us would understand what is meant by this. The best nomenclature would be to refer to a solution of “2% freshly depolymerized paraformaldehyde” assuming one started from the crystals/granules/powder formulation. Tom

Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Chair, MU Faculty Council
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/

From: Confocal Microscopy List [[hidden email]] On Behalf Of RICHARD BURRY
Sent: Thursday, March 05, 2009 9:33 AM
To: [hidden email]
Subject: Re: GFP/RFP quenching

Listers

I must jump in with an different view. Comments about the terms for formaldehyde fixation have been theoretical. However, when you see what people are doing in a core facility daily, the practical becomes more important! It would be nice if people doing immunocytochemistry or fixing GFP knew the difference between formalin and paraformaldehyde as a source of formaldehyde. However, users learning microscopy need different terms to describe the sources of formaldehyde. I recommend that rather than using formaldehyde, the terms formalin and paraformaldehyde be used to describe the fixative in articles. While this is not correct, the amount of time and money saved by people using the wrong chemical will justify being inaccurate.

Dick

----- Original Message -----
From: Steffen Dietzel <[hidden email]>
Date: Thursday, March 5, 2009 4:57 am
Subject: Re: GFP/RFP quenching
To: [hidden email]

>
> And while we are on nomenclature, a bit more wise cracking: there is no such thing as a
> paraformaldehyde solution. Paraformaldehyde is a polymer (white powder) that is unsoluble. Heat
> transforms it to the gas formaldehyde which can be solved in water.
>
> Cheers
>
> Steffen
>
> At 18:21 04.03.2009, you wrote:
> >Hi, Scott-
> >We have found that limiting the duration of
> >formaldehyde fixation to 10 minutes does
> >conserve the GFP signal. It seems to decline
> >the longer the cells sit in the fixative.
> >Carol Heckman
> >Center for Microscopy & Microanalysis
> >Bowling Green State University
> >________________________________________
> >From: Confocal Microscopy List
> >[[hidden email]] On Behalf Of
> >Kelly Lundsten [[hidden email]]
> >Sent: Wednesday, March 04, 2009 10:11 AM
> >To: [hidden email]
> >Subject: Re: GFP/RFP quenching
> >
> >Hi Scott,
> >
> >Any denaturant will be a potential problem for the
> protein. This
> >includes any MeOH that might be used to stabilize the
> PFA. Have them
> >make up fresh PFA the hard way, with heat and time, or buy it
> with a no
> >MeOH used guarantee. Also, if they are sealing their
> coverslips with
> >nail polish, stop that. Use VALAP
> (Vaseline:lanolin:paraffin) or
> >something else inert. The nail polish can seep under the
> coverslip over
> >time, denaturing the GFP. This sounds like the more
> likely culprit in
> >this instance if there is signal right after coverslipping but that
> >signal fades quickly over time. Or, use an antifade that
> cures, one
> >that doesn't require sealant. I believe Prolong Gold fits
> that criteria
> >(cures without dehydration around the edges of the
> coverslip). If the
> >problem persists after trying these potential solutions, then
> the PFA
> >might be changing the efficiency of the GFP due to its cross-linking
> >effect. Trying lower percent concentration of the PFA
> would be your
> >next step.
> >
> >Good luck,
> >Kelly Lundsten
> >
> >Sr. App Scientist
> >ART, Inc.
> >
> >-----Original Message-----
> >From: Confocal Microscopy List
> [[hidden email]]>On Behalf Of Scott Howell
> >Sent: Wednesday, March 04, 2009 6:52 AM
> >To: [hidden email]
> >Subject: GFP/RFP quenching
> >
> >List,
> >
> >We have had some issues here with a lab where the GFP/RFP
> brightness in
> >fixed cells has become extremely variable. Like night and day.
> Wondering>if it may be related to the pH of their
> paraformaldehyde fix? Does one
> >particular pH seem to work best?? Any other ideas? Thanks.
> >
> >Scott J. Howell, Ph.D.
> >Manager, Imaging Module
> >Visual Sciences Research Center
> >Case Western Reserve University
> >2085 Adelbert Rd.
> >Institute of Pathology Room 106
> >Cleveland, Ohio 44106
> >216-368-2300
> >http://www.case.edu/med/vsrc/
>
> --
> -----------------------------------------------------------------
> ----------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
>
> Mail room (for letters etc.):
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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
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