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4% PFA might have a high osmolarity but since it crosses the membrane, but its tonicity is another question that has been debated extensively over the years.
Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
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http://www.biotech.missouri.edu/mcc/
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Christophe Leterrier
Sent: Wednesday, November 07, 2012 6:36 AM
To: [log in to unmask]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C
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I agree that the message of the Schnell et al. paper is somewhat skewed to promote live-cell imaging instead of immunocytochemistry.
Also the fact that fixation can alter the distribution of proteins is hardly new, see the Burry book, or Brock et al. 1999 Cytometry part A 35(4), 353-362 (and I'm sure there are others).
However, there are some good data nuggets, including live imaging of what happens during fixation, and the fact that 4% PFA already has a very high osmolarity (~1300 mOsm), so that adding sucrose to adjust osmolarity isn't really necessary.
Cheers,
Christophe
On Wed, Nov 7, 2012 at 12:22 PM, Mark Cannell <[log in to unmask]> wrote:
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> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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>
> I think the paper by Schnell et al. shows that live cell imaging of
> constructs is NOT a gold standard at all. This is made clearer by
> inspection of the supp. images!. It seems to me that the major
> conclusion of their paper is that permeabilisation can remove soluble
> proteins? Hardly a revelation... As for the reliability of immunocytochemistry. I think we are all aware of the problems...
>
> Cheers
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