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)
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
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
> [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
| Free forum by Nabble | Edit this page |