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Thanks for the correction, I had read that sucrose was for osmolarity
balance and observed a lot of people don't use it when fixing cultured
cells, so I inferred this was the reason. Very happy to learn the real
reason why people use sucrose in their PFA fixative! This is why I
love this list.

Cheers,

Christophe

On Wed, Nov 7, 2012 at 2:50 PM, Lemasters, John J. <[log in to unmask]> wrote:
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> Formaldehyde and glutaraldehyde cross membranes essentially as readily as water, as well established over the years. Thus, fixatives must be osmotically balanced with sucrose, salines, or buffers with phosphate and arsenate (cacodylate) ignoring the contribution of the aldehydes to osmotic strength. Without this osmotic adjustment, cells and organelles (e.g., mitochondria) will swell.
>
> --
> John J. Lemasters, MD, PhD
> Professor and GlaxoSmithKline Distinguished Endowed Chair
> Director, Center for Cell Death, Injury & Regeneration
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> Medical University of South Carolina
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Christophe Leterrier
> Sent: Wednesday, November 07, 2012 7:36 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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>> 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