Re: aliquoting 4% formaldehyde

Posted by Mel Symeonides on
URL: http://confocal-microscopy-list.275.s1.nabble.com/aliquoting-4-formaldehyde-tp7585663p7585678.html

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Thanks for bringing that up, Mike.

Cell blebbing after formaldehyde fixation seems to be a widely ignored
fact among microscopists. Indeed, in some fields formaldehyde is used
specifically to induce blebbing! Many people won't run into it because
they might permeabilize their cells immediately after fixation for
antibody staining, and that (mostly) prevents the blebbing from being an
issue, but I'm sure that many people also must have tons of blebbing in
their samples that they either ignore or are not aware is a fixation
artifact. Yes, I know I'm generalizing. These papers go into this
phenomenon to some extent:

http://www.ncbi.nlm.nih.gov/pubmed/23184065
http://www.ncbi.nlm.nih.gov/pubmed/24649401

I deal with this by permeabilizing the cells even if that's not strictly
needed. If I'm using a lipohilic dye, most of which are washed out by
the usual permeabilization detergents (Triton X-100, Tween-20), I use
low concentrations of digitonin instead. That preserves the lipophilic
dye while also dealing with the blebs, though keep in mind that it is
selective for high-cholesterol membrane and you might need to take that
into account depending on the nature of your experiment. The exact
concentrations used should be optimized for different cell types.

As for the aliquoting issue, we buy 10 mL ampules of 32% aqueous
formaldehyde from Electron Microscopy Sciences (#15714), which we store
at room temperature. Upon opening, these keep fine at room temperature
and protected from light for a few weeks or even months. We dilute down
to 4% in PBS for each use.

Regards,
Mel




On 9/20/2016 10:47 AM, MODEL, MICHAEL wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
>
> Hi Doug,
>
> We, too, keep it frozen, that's what many sources recommend to prevent formation of formic acid. But I haven't seen much trouble when people keep it at room temperature. In any case, freezing cannot hurt, and the poor quality of your sample may have been due to something else. But even the freshest formaldehyde solution may cause cell shrinking, swelling or blebbing.
>
> Mike Model
>
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
> Sent: Tuesday, September 20, 2016 10:34 AM
> To: [hidden email]
> Subject: aliquoting 4% formaldehyde
>
> ***** To join, leave or search the confocal microscopy listserv, go to: http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy Post images on http://www.imgur.com and include the link in your posting. *****
> I was talking with a student yesterday about their cells which did not look particularly good under the microscope. The discussion eventually came around to fixation. The student told me that he made up some 4% formaldehyde fixative and then froze back (20 deg C) aliquots for later use. This was something he had seem on an online research forum. In my fairly lengthy experience I have never heard of this being done and I am suspicious that this is NOT a good idea. Thoughts?
>
> Doug
>
> ------------------------------------------------------------------------------------------
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> Dept. of Cellular & Molecular Medicine, University of Arizona
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--
Menelaos Symeonides
Post-Doctoral Associate, Thali Lab
Department of Microbiology and Molecular Genetics
University of Vermont
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: [phone]

--
Menelaos Symeonides
Post-Doctoral Associate, Thali Lab
Department of Microbiology and Molecular Genetics
University of Vermont
318 Stafford Hall
95 Carrigan Dr
Burlington, VT 05405
[hidden email]
Phone: [phone]