3.7% PFA permeabilize cells?

[Aromis Storm]

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Hello everyone:

I once heard some people discussing that fixation of cells with 3.7% PFA at
room temperature for 30mins already permeabilizes the cells. Does anyone of
you might have experience or opinion on this comment?

Thanks a lot for your input in advance.

Regards,

Aro

[lechristophe]

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Dear Aro,

In my experience PFA 4% for 15 minutes will permeabilize cultured
hippocampal neurons to a certain extent (and this will vary from one cell
to another). It is very easy to check that in your model by using an
antibody against an intracellular epitope in one of your immuno channels,
without further permeabilization.

Regards,

Christophe


On Tue, Apr 2, 2013 at 5:04 PM, Aromis Storm <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello everyone:
>
> I once heard some people discussing that fixation of cells with 3.7% PFA at
> room temperature for 30mins already permeabilizes the cells. Does anyone of
> you might have experience or opinion on this comment?
>
> Thanks a lot for your input in advance.
>
> Regards,
>
> Aro
>

[Kate Luby-Phelps]

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If you use a 1:10 dilution of PFA from a 37% bottle, your fixative likely will
contain methanol, which permeabilizes the cells at room temperature. You may
not make holes big enough for antibodies, but personal experience tells me that
phalloidin can definitely get in. Unless you want to permeablize the cells, the
better way to do it is to make the PFA fresh from powder (laborious) or a
sealed ampule of 8 or 16% (expensive).

[Stephen Firth (Med)]

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Making PFA should not be too laborious, it's not very soluble at room
temperature, but dissolves immediately at 50 degrees C. I heat my PBS in a
microwave to about 50 and dissolve in a fume hood, filter through filter
paper and cool on ice. From powder to ready solution in 10 to 15 min.
Cheers,
Stephen


On 3 April 2013 16:15, Kate Luby-Phelps <[hidden email]>wrote:

> *****
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> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you use a 1:10 dilution of PFA from a 37% bottle, your fixative likely
> will
> contain methanol, which permeabilizes the cells at room temperature. You
> may
> not make holes big enough for antibodies, but personal experience tells me
> that
> phalloidin can definitely get in. Unless you want to permeablize the
> cells, the
> better way to do it is to make the PFA fresh from powder (laborious) or a
> sealed ampule of 8 or 16% (expensive).
>

--
Stephen Firth
Manager of Advanced Optical Microscopy
Monash Micro Imaging
Monash University

[Michał Majkowski]

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Hallo,
I am working with 3.7% PFA as a fixative (1:10 from 37% PFA solution
containing methanol as a stabilizer). After 10 min. of fixation holes
made by ME-OH are big enough to allow Cholera Toxin B subunit get in
(CT-B+ fluorophore ~ 12 kDa). I have no experience with IgG (~ 150 kDa).
best,
Michal

[Masur, Sandra]

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Even freshly made 3.7% p-formaldehyde fixation at room temperature for 30 min will permeabilize mammalian cells to phalloidin. We found that fixation on ice was the only way to prevent the permeabilization.

On Apr 3, 2013, at 1:15 AM, Kate Luby-Phelps wrote:

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If you use a 1:10 dilution of PFA from a 37% bottle, your fixative likely will
contain methanol, which permeabilizes the cells at room temperature. You may
not make holes big enough for antibodies, but personal experience tells me that
phalloidin can definitely get in. Unless you want to permeablize the cells, the
better way to do it is to make the PFA fresh from powder (laborious) or a
sealed ampule of 8 or 16% (expensive).

[lechristophe]

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Aro,

you can also have a look at that previous discussion on the list:
http://confocal-microscopy-list.588098.n2.nabble.com/Paraformaldehyde-and-permeabilization-td7578873.html


On Wed, Apr 3, 2013 at 1:12 PM, Masur, Sandra <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Even freshly made 3.7% p-formaldehyde fixation at room temperature for 30
> min will permeabilize mammalian cells to phalloidin. We found that fixation
> on ice was the only way to prevent the permeabilization.
>
> On Apr 3, 2013, at 1:15 AM, Kate Luby-Phelps wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you use a 1:10 dilution of PFA from a 37% bottle, your fixative likely
> will
> contain methanol, which permeabilizes the cells at room temperature. You
> may
> not make holes big enough for antibodies, but personal experience tells me
> that
> phalloidin can definitely get in. Unless you want to permeablize the
> cells, the
> better way to do it is to make the PFA fresh from powder (laborious) or a
> sealed ampule of 8 or 16% (expensive).
>

[Aromis Storm]

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Thank you for all your input and the link shared by Christophe is indeed
very helpful: this seems to be an old topic which is likely to come up again
in the future.

I got my IF images now and I can share some fresh personal experience. 3.7%
PFA (made from power, stored and aliquot in -20C, one time use) fixation of
HeLa cells at room temperature for 30mins allows detection of TNPO3 with
antibodies.

One distinctive phenomenon is that ,compared with cells that have been
permeabilized by Triton, the cells that were treated only with PFA showed
much stronger auto-fluorescence in the 488 channel. Is this because some
auto-fluorescence 'sources' were not washed out of the cells because the
cells were not permeabilized enough?

Greetings,

Aro

-----邮件原件-----
发件人: Confocal Microscopy List [mailto:[hidden email]]
代表 Christophe Leterrier
发送时间: Mittwoch, 3. April 2013 13:54
收件人: [hidden email]
主题: Re: 3.7% PFA permeabilize cells?

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Aro,

you can also have a look at that previous discussion on the list:
http://confocal-microscopy-list.588098.n2.nabble.com/Paraformaldehyde-and-pe
rmeabilization-td7578873.html


On Wed, Apr 3, 2013 at 1:12 PM, Masur, Sandra <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Even freshly made 3.7% p-formaldehyde fixation at room temperature for
> 30 min will permeabilize mammalian cells to phalloidin. We found that
> fixation on ice was the only way to prevent the permeabilization.
>
> On Apr 3, 2013, at 1:15 AM, Kate Luby-Phelps wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> If you use a 1:10 dilution of PFA from a 37% bottle, your fixative
> likely will contain methanol, which permeabilizes the cells at room
> temperature. You may not make holes big enough for antibodies, but
> personal experience tells me that phalloidin can definitely get in.
> Unless you want to permeablize the cells, the better way to do it is
> to make the PFA fresh from powder (laborious) or a sealed ampule of 8
> or 16% (expensive).
>

[Martin Wessendorf-2]

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On 4/4/2013 3:55 PM, Aromis wrote:

> One distinctive phenomenon is that ,compared with cells that have been
> permeabilized by Triton, the cells that were treated only with PFA showed
> much stronger auto-fluorescence in the 488 channel. Is this because some
> auto-fluorescence 'sources' were not washed out of the cells because the
> cells were not permeabilized enough?

The increased autofluorescence may simply be due to formaldehyde (--or
other aldehyde fixatives) reacting with primary amines to form
autofluorescent Schiff bases.  If you reduce these chemically using
NaBH4, you'll reduce the autofluorescence.

There are a number of references on this; here's one: Clancy B. Cauller
LJ., "Reduction of background autofluorescence in brain sections
following immersion in sodium borohydride."  Journal of Neuroscience
Methods. 83(2):97-102, 1998

Good luck!

Martin Wessendorf
--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Knecht, David]

I am very confused by your observations.  My experience is that small molecules get through the membranes of fixed cells, but antibodies do not and you must use Triton et al. to stain intracellular compartments.  We routinely do phagocytosis assays with coated particles and the basis of the assay is that you fix with formaldyhyde without triton and then antibody stain for the protein coating the particle.  The particles on the outside of the cell become labelled and the ones on the inside are not labeled.  The assay would not work if the formaldehyde alone permeabilized the cells.  We generally use 1-2% formaldehyde and only for about 10 minutes.  Also, we do not see any significant increase in fluorescence at 488 when you fix with formaldehyde alone, only when you use glutaraldehyde.  That autofluorescence is to some extent reduced by borohydride, but not much in our hands so we keep the glut at 0.1% which does not cause enough fluorescence to worry about using borohydride.  I guess it is possible the long fixation with more formaldehyde is doing more damage the cell membranes.  Also, our particles, because of phagocytosis, are inside the phagosomal membrane so the antibody would have to cross two membranes and perhaps the phagosomal membrane is less permeant after fixation. Is your fluorescence localized to the nucleus?  Dave

On Apr 4, 2013, at 4:55 PM, Aromis wrote:

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Thank you for all your input and the link shared by Christophe is indeed
very helpful: this seems to be an old topic which is likely to come up again
in the future.

I got my IF images now and I can share some fresh personal experience. 3.7%
PFA (made from power, stored and aliquot in -20C, one time use) fixation of
HeLa cells at room temperature for 30mins allows detection of TNPO3 with
antibodies.

One distinctive phenomenon is that ,compared with cells that have been
permeabilized by Triton, the cells that were treated only with PFA showed
much stronger auto-fluorescence in the 488 channel. Is this because some
auto-fluorescence 'sources' were not washed out of the cells because the
cells were not permeabilized enough?

Greetings,

Aro

-----邮件原件-----
发件人: Confocal Microscopy List [mailto:[hidden email]]
代表 Christophe Leterrier
发送时间: Mittwoch, 3. April 2013 13:54
收件人: [hidden email]
主题: Re: 3.7% PFA permeabilize cells?

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Aro,

you can also have a look at that previous discussion on the list:
http://confocal-microscopy-list.588098.n2.nabble.com/Paraformaldehyde-and-pe
rmeabilization-td7578873.html


On Wed, Apr 3, 2013 at 1:12 PM, Masur, Sandra <[hidden email]> wrote:

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Even freshly made 3.7% p-formaldehyde fixation at room temperature for
30 min will permeabilize mammalian cells to phalloidin. We found that
fixation on ice was the only way to prevent the permeabilization.

On Apr 3, 2013, at 1:15 AM, Kate Luby-Phelps wrote:

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If you use a 1:10 dilution of PFA from a 37% bottle, your fixative
likely will contain methanol, which permeabilizes the cells at room
temperature. You may not make holes big enough for antibodies, but
personal experience tells me that phalloidin can definitely get in.
Unless you want to permeablize the cells, the better way to do it is
to make the PFA fresh from powder (laborious) or a sealed ampule of 8
or 16% (expensive).


David Knecht, Ph.D.
Professor and Head of Microscopy Facility
Department of Molecular and Cell Biology
U-3125
91 N. Eagleville Rd.
University of Connecticut
Storrs, CT 06269
860-486-2200
860-486-4331 (fax)

[Ellen T. Arena]

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I also find that the buffer you use for dilution of the PFA can also make a difference.  I use a phosphate buffer (pH7.4) - for 100ml use 77.4 mL Na2HPO4 (1M) and 22.6 mL NaH2PO4 (1M).  Apparently - it is less harsh to the membrane without potassium present.  Also - I follow fixation with an incubation in 50 mM NH4Cl - this helps stop the fixation and therefore the continued permeabilization.

Hope it helps!

Eleln




On 3 avr. 2013, at 07:34, Stephen Firth wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Making PFA should not be too laborious, it's not very soluble at room
> temperature, but dissolves immediately at 50 degrees C. I heat my PBS in a
> microwave to about 50 and dissolve in a fume hood, filter through filter
> paper and cool on ice. From powder to ready solution in 10 to 15 min.
> Cheers,
> Stephen
>
>
> On 3 April 2013 16:15, Kate Luby-Phelps <[hidden email]>wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> If you use a 1:10 dilution of PFA from a 37% bottle, your fixative likely
>> will
>> contain methanol, which permeabilizes the cells at room temperature. You
>> may
>> not make holes big enough for antibodies, but personal experience tells me
>> that
>> phalloidin can definitely get in. Unless you want to permeablize the
>> cells, the
>> better way to do it is to make the PFA fresh from powder (laborious) or a
>> sealed ampule of 8 or 16% (expensive).
>>
>
>
>
> --
> Stephen Firth
> Manager of Advanced Optical Microscopy
> Monash Micro Imaging
> Monash University

------------------------------------------------------
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Tel: [phone]
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