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
[log in to unmask] 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)
> [log in to unmask]
>
> http://www.biology.missouri.edu/faculty/phillips.html
> http://www.biotech.missouri.edu/mcc/
>
> From: Confocal Microscopy List [mailto:[log in to unmask]
> ] On Behalf Of RICHARD BURRY
> Sent: Thursday, March 05, 2009 9:33 AM
> To: [log in to unmask]
> 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 <[log in to unmask]>
> Date: Thursday, March 5, 2009 4:57 am
> Subject: Re: GFP/RFP quenching
> To: [log in to unmask]
>
>
> >
> > 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
> > >[[log in to unmask]] On Behalf Of
> > >Kelly Lundsten [[log in to unmask]]
> > >Sent: Wednesday, March 04, 2009 10:11 AM
> > >To: [log in to unmask]
> > >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:[log in to unmask]]>On Behalf Of Scott Howell
> > >Sent: Wednesday, March 04, 2009 6:52 AM
> > >To: [log in to unmask]
> > >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)
> >
> >
> > --
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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
> 277 Biomedical Research Tower
> 460 West Twelfth Avenue
> Columbus, Ohio 43210
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