Why perform Methanol, Methanol/Acetone fixation at -20C

[Ke Peng]

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Hello Everyone:

Sorry to bother you with a naive/entry level question: why the fixation with
methanol or methanol/acetone are normally performed at -20C? I tried to look
it up on the internet but except for finding a number of protocols
describing this I have not found one explaining this so far.

Thanks a lot for your help in advance.

Ke

[Guy Cox-2]

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These are extremely strong solvents.  To extract enough lipids to make cells permeable we may need to use them but unless we do it at low temperature (slowing down the reaction) we will end up with nothing left.

                                                                           Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of aropro
Sent: Tuesday, 6 November 2012 10:52 PM
To: [hidden email]
Subject: Why perform Methanol, Methanol/Acetone fixation at -20C

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Hello Everyone:

Sorry to bother you with a naive/entry level question: why the fixation with methanol or methanol/acetone are normally performed at -20C? I tried to look it up on the internet but except for finding a number of protocols describing this I have not found one explaining this so far.

Thanks a lot for your help in advance.

Ke

[Paul Rigby-2]

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Hi Ke,
What a great question!! Why don't more of us (biologists/microscopists etc) ever question the basic protocols that are in common use. Often these protocols are the result of "chinese wispers" and are passed down and modified over time. Eventually, either something stops working or someone like yourself actually thinks "outside the box". Well done.

Now to try to answer your question - methanol or methanol/acetone are coagulative fixatives most commonly used to preserve cytoskeletal elements in cells. Fixation is usually for a short time (5 or 10 minutes at -20C). It seems they do a "better" job at preserving or exposing some epitopes, allowing better antibody access than do cross-linking fixatives like formaldehyde or glutaraldehyde. Why? - I have no idea; they just seem to work better in some cases. As Guy has suggested, perhaps it is related to the strong lipid dissolving properties.

As an aside, if one looks at the chemistry literature for aldehyde fixation, it is interesting that for complete fixation and stabilisation, times significantly longer than 24 hours are required for the cross linking reactions to go to completion. (For a good explanation see Kiernan, J.A., Microscopy Today, January 2000, pp 8-12.). So, why do we typically fix single cells for only 10 or 30 minutes in formaldehyde solutions before immunolabelling? Probably because it works. Do longer fixation times (24 hours or more) work better for preservation of single cell structures prior to immunolabelling? I suspect not, but does anyone have direct experience?

Cheers
Paul

Assoc. Prof. Paul Rigby
Centre for Microscopy, Characterisation & Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6007
Australia

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of aropro
Sent: Tuesday, 6 November 2012 7:52 PM
To: [hidden email]
Subject: Why perform Methanol, Methanol/Acetone fixation at -20C

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Hello Everyone:

Sorry to bother you with a naive/entry level question: why the fixation with methanol or methanol/acetone are normally performed at -20C? I tried to look it up on the internet but except for finding a number of protocols describing this I have not found one explaining this so far.

Thanks a lot for your help in advance.

Ke

[lechristophe]

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Something related to this non-complete fixation by aldehydes: Akihiro
Kusumi's lab reported two years ago that lipids and membrane proteins
were still diffusing in the plasma membrane after fixation for up to
90 mintues in 4% PFA. Cold methanol was better in this regard,
although a residual mobility was still observed:
Tanaka, K. A. K., Suzuki, K. G. N., Shirai, Y. M., Shibutani, S. T.,
Miyahara, M. S. H., Tsuboi, H., et al. (2010). Membrane molecules
mobile even after chemical fixation. Nature Methods.
doi:10.1038/nmeth.f.314


Another good & recent article about immunocytochemistry procedures has
been published in Nature Methods:
Schnell, U., Dijk, F., Sjollema, K. A., & Giepmans, B. N. G. (2012).
Immunolabeling artifacts and the need for live-cell imaging. Nature
Methods, 9(2), 152–158. doi:10.1038/nmeth.1855


And I highly suggest to people that are doing
immunocyto/histochemistry to read R. Burry's book. It covers
everything from fixation to fluorescence imaging and explains a lot of
"chinese whispers", debunking a few ones on the way:
Burry, R. W. (2010). Immunocytochemistry. Springer Verlag.
http://books.google.fr/books?id=svzyJdQVsaEC&printsec=frontcover&hl=fr&source=gbs_ge_summary_r&cad=0#v=onepage&q&f=false

Cheers,

--
Christophe Leterrier
Researcher
Axonal Domains Architecture Team
CRN2M CNRS UMR 7286
Aix Marseille University, France


On Wed, Nov 7, 2012 at 6:33 AM, Paul Rigby <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
>
> As an aside, if one looks at the chemistry literature for aldehyde fixation, it is interesting that for complete fixation and stabilisation, times significantly longer than 24 hours are required for the cross linking reactions to go to completion. (For a good explanation see Kiernan, J.A., Microscopy Today, January 2000, pp 8-12.). So, why do we typically fix single cells for only 10 or 30 minutes in formaldehyde solutions before immunolabelling? Probably because it works. Do longer fixation times (24 hours or more) work better for preservation of single cell structures prior to immunolabelling? I suspect not, but does anyone have direct experience?
>

[xavier Sanjuan]

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Years ago I worked with vascular tissue; we routinely fixed arterial rings
of aprox. 2 mm overnight at the 4º room with 4% PFA, but sometimes we left
the rings in PFA one even two days without a signifficant effect on the
result of the immunostaining. I accidentally forgot a set of arterial rings
for about a month in the fixative (for sure PFA was gradually degraded over
that time)... And the vessels turned into a hard piece of cement totally
impermeable to antibodies.

You can always learn something from mistakes!

Best,

Xavi.

PS: Thanks Christophe for pointing at Burry's book, seems the Bible of
immunolabeling!
___________________________________

*Xavier Sanjuan**
*Advanced Light Microscopy Unit
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88
08003 Barcelona - Spain
Tel: + 34 93 316 0206
Fax: + 34 93 316 09 01
E-mail: [hidden email]
Web:
http://pasteur.crg.es/portal/page/portal/Internet/03_CORES/Core_Facilities/Advanced%20Light%20Microscopy%20Unit


2012/11/7 Paul Rigby <[hidden email]>

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Ke,
> What a great question!! Why don't more of us (biologists/microscopists
> etc) ever question the basic protocols that are in common use. Often these
> protocols are the result of "chinese wispers" and are passed down and
> modified over time. Eventually, either something stops working or someone
> like yourself actually thinks "outside the box". Well done.
>
> Now to try to answer your question - methanol or methanol/acetone are
> coagulative fixatives most commonly used to preserve cytoskeletal elements
> in cells. Fixation is usually for a short time (5 or 10 minutes at -20C).
> It seems they do a "better" job at preserving or exposing some epitopes,
> allowing better antibody access than do cross-linking fixatives like
> formaldehyde or glutaraldehyde. Why? - I have no idea; they just seem to
> work better in some cases. As Guy has suggested, perhaps it is related to
> the strong lipid dissolving properties.
>
> As an aside, if one looks at the chemistry literature for aldehyde
> fixation, it is interesting that for complete fixation and stabilisation,
> times significantly longer than 24 hours are required for the cross linking
> reactions to go to completion. (For a good explanation see Kiernan, J.A.,
> Microscopy Today, January 2000, pp 8-12.). So, why do we typically fix
> single cells for only 10 or 30 minutes in formaldehyde solutions before
> immunolabelling? Probably because it works. Do longer fixation times (24
> hours or more) work better for preservation of single cell structures prior
> to immunolabelling? I suspect not, but does anyone have direct experience?
>
> Cheers
> Paul
>
> Assoc. Prof. Paul Rigby
> Centre for Microscopy, Characterisation & Analysis (M510)
> The University of Western Australia
> 35 Stirling Highway
> Crawley  WA  6007
> Australia
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of aropro
> Sent: Tuesday, 6 November 2012 7:52 PM
> To: [hidden email]
> Subject: Why perform Methanol, Methanol/Acetone fixation at -20C
>
>  *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Everyone:
>
> Sorry to bother you with a naive/entry level question: why the fixation
> with methanol or methanol/acetone are normally performed at -20C? I tried
> to look it up on the internet but except for finding a number of protocols
> describing this I have not found one explaining this so far.
>
> Thanks a lot for your help in advance.
>
> Ke
>

[Mark Cannell-2]

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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


On 7/11/2012, at 8:37 AM, Christophe Leterrier <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Something related to this non-complete fixation by aldehydes: Akihiro
> Kusumi's lab reported two years ago that lipids and membrane proteins
> were still diffusing in the plasma membrane after fixation for up to
> 90 mintues in 4% PFA. Cold methanol was better in this regard,
> although a residual mobility was still observed:
> Tanaka, K. A. K., Suzuki, K. G. N., Shirai, Y. M., Shibutani, S. T.,
> Miyahara, M. S. H., Tsuboi, H., et al. (2010). Membrane molecules
> mobile even after chemical fixation. Nature Methods.
> doi:10.1038/nmeth.f.314
>
>
> Another good & recent article about immunocytochemistry procedures has
> been published in Nature Methods:
> Schnell, U., Dijk, F., Sjollema, K. A., & Giepmans, B. N. G. (2012).
> Immunolabeling artifacts and the need for live-cell imaging. Nature
> Methods, 9(2), 152–158. doi:10.1038/nmeth.1855
>
>
> And I highly suggest to people that are doing
> immunocyto/histochemistry to read R. Burry's book. It covers
> everything from fixation to fluorescence imaging and explains a lot of
> "chinese whispers", debunking a few ones on the way:
> Burry, R. W. (2010). Immunocytochemistry. Springer Verlag.
> http://books.google.fr/books?id=svzyJdQVsaEC&printsec=frontcover&hl=fr&source=gbs_ge_summary_r&cad=0#v=onepage&q&f=false
>
> Cheers,
>
> --
> Christophe Leterrier
> Researcher
> Axonal Domains Architecture Team
> CRN2M CNRS UMR 7286
> Aix Marseille University, France
>
>
> On Wed, Nov 7, 2012 at 6:33 AM, Paul Rigby <[hidden email]> wrote:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>>
>> As an aside, if one looks at the chemistry literature for aldehyde fixation, it is interesting that for complete fixation and stabilisation, times significantly longer than 24 hours are required for the cross linking reactions to go to completion. (For a good explanation see Kiernan, J.A., Microscopy Today, January 2000, pp 8-12.). So, why do we typically fix single cells for only 10 or 30 minutes in formaldehyde solutions before immunolabelling? Probably because it works. Do longer fixation times (24 hours or more) work better for preservation of single cell structures prior to immunolabelling? I suspect not, but does anyone have direct experience?
>>

Mark  B. Cannell Ph.D. FRSNZ
Professor of Cardiac Cell Biology
School of Physiology&  Pharmacology
Medical Sciences Building
University of Bristol
Bristol
BS8 1TD UK

[hidden email]

[lechristophe]

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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
<[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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

[Phillips, Thomas E.]

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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
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Wednesday, November 07, 2012 6:36 AM
To: [hidden email]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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 <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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

[Lemasters, John J.]

*****
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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
Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
DD504 Drug Discovery Building
70 President Street, MSC 140
Charleston, SC 29425

Office: 843-876-2360
Lab: 843-876-2354
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Wednesday, November 07, 2012 7:36 AM
To: [hidden email]
Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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 <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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

[lechristophe]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

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. <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> Departments of Drug Discovery & Biomedical Sciences and Biochemistry & Molecular Biology
> Medical University of South Carolina
> DD504 Drug Discovery Building
> 70 President Street, MSC 140
> Charleston, SC 29425
>
> Office: 843-876-2360
> Lab: 843-876-2354
> Fax: [phone]
> Email: [hidden email]
> http://academicdepartments.musc.edu/ccdir
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
> Sent: Wednesday, November 07, 2012 7:36 AM
> To: [hidden email]
> Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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 <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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

[Patrick Van Oostveldt]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear,

The use of methanol/aceton at minus 20 is infact a simple  
cryosubstitution approach. The mixture should be water free and used  
in great volume. If e.g. using a mixture that was out of the freezer  
for some times and put back in the freezer will give bad tubuline  
fixation because it attracts water on the bench and if the bottle is  
not filled, even more water from the air will dissolve in the mixture.  
A solution to this is to include some water attracting resins in the  
solute, this will bind the water and you fixative will stay "water  
free".
Changing from -20 to room temperature will disolve lipids but at that  
moment the proteins are nearly completely dehydrated and will stay in  
a good or natural shape.

Patrick
--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel at lab: 09 264 5969
priv tel: [phone]
fax [phone]
GSM [phone]



Citeren Christophe Leterrier <[hidden email]>:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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. <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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
>> Departments of Drug Discovery & Biomedical Sciences and  
>> Biochemistry & Molecular Biology
>> Medical University of South Carolina
>> DD504 Drug Discovery Building
>> 70 President Street, MSC 140
>> Charleston, SC 29425
>>
>> Office: 843-876-2360
>> Lab: 843-876-2354
>> Fax: [phone]
>> Email: [hidden email]
>> http://academicdepartments.musc.edu/ccdir
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List  
>> [mailto:[hidden email]] On Behalf Of Christophe  
>> Leterrier
>> Sent: Wednesday, November 07, 2012 7:36 AM
>> To: [hidden email]
>> Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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  
>> <[hidden email]> wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> 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

[Steffen Dietzel]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

On 07.11.2012 13:35, Christophe Leterrier wrote:
> 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.


A couple of years ago, we did some similar comparisons, before and after
formaldehyde fixation (and after FISH) for chromosomal sites labeled
with GFP, anti-GFP antibodies and FISH probes. We were actually
surprised how well the various methods agreed with each other (although
of course results were not identical). In a related paper we compared
various fixation methods for cell nuclei, variations of formaldehyde and
methanol-acetic acid fixation. Both papers are open access, in case your
interested (see below for the links).

While on the topic: is anybody aware of live/dead comparisons after a
zinc fixation protocol? I didn't find much about that so far.

Steffen


BMC Biotechnol. 2007 Dec 20;7:92.
Quantitative comparison of DNA detection by GFP-lac repressor tagging,
fluorescence in situ hybridization and immunostaining.
Kim IH, Nagel J, Otten S, Knerr B, Eils R, Rohr K, Dietzel S.
https://www.biomedcentral.com/1472-6750/7/92

and
Chromosoma. 2007 Apr;116(2):117-33. Epub 2006 Nov 22.
Preservation of large-scale chromatin structure in FISH experiments.
Hepperger C, Otten S, von Hase J, Dietzel S.
http://link.springer.com/article/10.1007%2Fs00412-006-0084-2

>
> Cheers,
>
> Christophe
>

--
------------------------------------------------------------
Steffen Dietzel, PD Dr. rer. nat
Ludwig-Maximilians-Universität München
Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
Head of light microscopy

Mail room:
Marchioninistr. 15, D-81377 München

Building location:
Marchioninistr. 27,  München-Großhadern

[lechristophe]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Hi,

I was reading again this great thread and regarding methanol fixation
of cells, I'd like to go back to Patrick's comments.

Could you or someone else elaborate on what kind of "water-attracting
resin" you can put in the methanol to avoid water contamination? What
is it and where do you find it?

Second, I'm not sure I understand the last phrase about changing the
temperature from -20°C to room temperature:

"Changing from -20 to room temperature will disolve lipids but at that
moment the proteins are nearly completely dehydrated and will stay in
a good or natural shape."

Do you mean it is important that the MeOH is at -20°C at moment you
put it on cells, and then you can let it warm on the bench during the
~5 minutes fixation? I usually put the cells in cold MeOH in the
freezer during the 5 minutes but I've seen people doing it at RT, so
what would be the best to do?

Thanks for your advices,

Christophe

On Wed, Nov 7, 2012 at 4:01 PM, Patric Van Oostveldt
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear,
>
> The use of methanol/aceton at minus 20 is infact a simple cryosubstitution
> approach. The mixture should be water free and used in great volume. If e.g.
> using a mixture that was out of the freezer for some times and put back in
> the freezer will give bad tubuline fixation because it attracts water on the
> bench and if the bottle is not filled, even more water from the air will
> dissolve in the mixture. A solution to this is to include some water
> attracting resins in the solute, this will bind the water and you fixative
> will stay "water free".
> Changing from -20 to room temperature will disolve lipids but at that moment
> the proteins are nearly completely dehydrated and will stay in a good or
> natural shape.
>
> Patrick
> --
> Dep. Moleculaire Biotechnologie
> Coupure links 653
> B 9000 GENT
>
> tel at lab: 09 264 5969
> priv tel: [phone]
> fax [phone]
> GSM [phone]
>
>
>
> Citeren Christophe Leterrier <[hidden email]>:
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> 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. <[hidden email]>
>> wrote:
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> 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
>>> Departments of Drug Discovery & Biomedical Sciences and Biochemistry &
>>> Molecular Biology
>>> Medical University of South Carolina
>>> DD504 Drug Discovery Building
>>> 70 President Street, MSC 140
>>> Charleston, SC 29425
>>>
>>> Office: 843-876-2360
>>> Lab: 843-876-2354
>>> Fax: [phone]
>>> Email: [hidden email]
>>> http://academicdepartments.musc.edu/ccdir
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>> On Behalf Of Christophe Leterrier
>>> Sent: Wednesday, November 07, 2012 7:36 AM
>>> To: [hidden email]
>>> Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> 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
>>> <[hidden email]> wrote:
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> 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

[lechristophe]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

and one last question: I've seen people putting EGTA or EGTA/MgCl2
(MES buffer) in the methanol for fixation. What is the rationale
behing that?

http://www.microscopy.med.ualberta.ca/techniques/cell-fixation-with-ice-cold-methanol/

What are the advantages compared to pure methanol, specifically does
it contradict the water-free requirement as you'll have to introduce
some water in the mixture when adding EGTA or MgCl2 ?

Thanks again,

Christophe

On Thu, Nov 22, 2012 at 11:51 AM, Christophe Leterrier
<[hidden email]> wrote:

> Hi,
>
> I was reading again this great thread and regarding methanol fixation
> of cells, I'd like to go back to Patrick's comments.
>
> Could you or someone else elaborate on what kind of "water-attracting
> resin" you can put in the methanol to avoid water contamination? What
> is it and where do you find it?
>
> Second, I'm not sure I understand the last phrase about changing the
> temperature from -20°C to room temperature:
>
> "Changing from -20 to room temperature will disolve lipids but at that
> moment the proteins are nearly completely dehydrated and will stay in
> a good or natural shape."
>
> Do you mean it is important that the MeOH is at -20°C at moment you
> put it on cells, and then you can let it warm on the bench during the
> ~5 minutes fixation? I usually put the cells in cold MeOH in the
> freezer during the 5 minutes but I've seen people doing it at RT, so
> what would be the best to do?
>
> Thanks for your advices,
>
> Christophe
>
> On Wed, Nov 7, 2012 at 4:01 PM, Patric Van Oostveldt
> <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear,
>>
>> The use of methanol/aceton at minus 20 is infact a simple cryosubstitution
>> approach. The mixture should be water free and used in great volume. If e.g.
>> using a mixture that was out of the freezer for some times and put back in
>> the freezer will give bad tubuline fixation because it attracts water on the
>> bench and if the bottle is not filled, even more water from the air will
>> dissolve in the mixture. A solution to this is to include some water
>> attracting resins in the solute, this will bind the water and you fixative
>> will stay "water free".
>> Changing from -20 to room temperature will disolve lipids but at that moment
>> the proteins are nearly completely dehydrated and will stay in a good or
>> natural shape.
>>
>> Patrick
>> --
>> Dep. Moleculaire Biotechnologie
>> Coupure links 653
>> B 9000 GENT
>>
>> tel at lab: 09 264 5969
>> priv tel: [phone]
>> fax [phone]
>> GSM [phone]
>>
>>
>>
>> Citeren Christophe Leterrier <[hidden email]>:
>>
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> 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. <[hidden email]>
>>> wrote:
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> 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
>>>> Departments of Drug Discovery & Biomedical Sciences and Biochemistry &
>>>> Molecular Biology
>>>> Medical University of South Carolina
>>>> DD504 Drug Discovery Building
>>>> 70 President Street, MSC 140
>>>> Charleston, SC 29425
>>>>
>>>> Office: 843-876-2360
>>>> Lab: 843-876-2354
>>>> Fax: [phone]
>>>> Email: [hidden email]
>>>> http://academicdepartments.musc.edu/ccdir
>>>>
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>>> On Behalf Of Christophe Leterrier
>>>> Sent: Wednesday, November 07, 2012 7:36 AM
>>>> To: [hidden email]
>>>> Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> 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
>>>> <[hidden email]> wrote:
>>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> 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

[Patrick Van Oostveldt]

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Dear,

The product used for water absorbance is so called "molecular sieves"
This in fact is a well defined zeolite.
A website for some information:
http://www.zeochem.com/en/products/applications/alcohol_deydration.htm 
or google for "Webest Molecular Sieves"

You can expect it is more effective for ethanol than for methanol, it  
works also with methanol because the amount of water that has to be  
eliminated from methanol is far less than for ethanol.

About the last paragraph I use methanol cooled in the freezer (-20  
celsius), but a minus 80 is also possible.
The fairly large amount of a water free solvent will nearly instantly  
dehydrate the tissue comparable to cryosubstitution if immersing the  
slide. If the whole is "dry", temperature will not have a bad effect.  
But if you use small amounts of absolute ethanol or methanol the risk  
is real that you will have to much water.

I use in general a coplin jar (50ml) for one or two slides. After  
fixation (up to 5 min) I transfer the jar to the bench and start  
staining with water based reagents after temperature equilibration.  
Aceton can also be used but aceton is less well mixed with water. If  
small amounts of aceton stay trapped in the tissue it can interfer  
with the water solvents.

Hope this information is usefull.

Bye

Patrick


--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel at lab: 09 264 5969
priv tel: [phone]
fax [phone]
GSM [phone]



Citeren Christophe Leterrier <[hidden email]>:

> Hi,
>
> I was reading again this great thread and regarding methanol fixation
> of cells, I'd like to go back to Patrick's comments.
>
> Could you or someone else elaborate on what kind of "water-attracting
> resin" you can put in the methanol to avoid water contamination? What
> is it and where do you find it?
>
> Second, I'm not sure I understand the last phrase about changing the
> temperature from -20°C to room temperature:
>
> "Changing from -20 to room temperature will disolve lipids but at that
> moment the proteins are nearly completely dehydrated and will stay in
> a good or natural shape."
>
> Do you mean it is important that the MeOH is at -20°C at moment you
> put it on cells, and then you can let it warm on the bench during the
> ~5 minutes fixation? I usually put the cells in cold MeOH in the
> freezer during the 5 minutes but I've seen people doing it at RT, so
> what would be the best to do?
>
> Thanks for your advices,
>
> Christophe
>
> On Wed, Nov 7, 2012 at 4:01 PM, Patric Van Oostveldt
> <[hidden email]> wrote:
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear,
>>
>> The use of methanol/aceton at minus 20 is infact a simple cryosubstitution
>> approach. The mixture should be water free and used in great volume. If e.g.
>> using a mixture that was out of the freezer for some times and put back in
>> the freezer will give bad tubuline fixation because it attracts water on the
>> bench and if the bottle is not filled, even more water from the air will
>> dissolve in the mixture. A solution to this is to include some water
>> attracting resins in the solute, this will bind the water and you fixative
>> will stay "water free".
>> Changing from -20 to room temperature will disolve lipids but at that moment
>> the proteins are nearly completely dehydrated and will stay in a good or
>> natural shape.
>>
>> Patrick
>> --
>> Dep. Moleculaire Biotechnologie
>> Coupure links 653
>> B 9000 GENT
>>
>> tel at lab: 09 264 5969
>> priv tel: [phone]
>> fax [phone]
>> GSM [phone]
>>
>>
>>
>> Citeren Christophe Leterrier <[hidden email]>:
>>
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> 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. <[hidden email]>
>>> wrote:
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> 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
>>>> Departments of Drug Discovery & Biomedical Sciences and Biochemistry &
>>>> Molecular Biology
>>>> Medical University of South Carolina
>>>> DD504 Drug Discovery Building
>>>> 70 President Street, MSC 140
>>>> Charleston, SC 29425
>>>>
>>>> Office: 843-876-2360
>>>> Lab: 843-876-2354
>>>> Fax: [phone]
>>>> Email: [hidden email]
>>>> http://academicdepartments.musc.edu/ccdir
>>>>
>>>>
>>>> -----Original Message-----
>>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>>> On Behalf Of Christophe Leterrier
>>>> Sent: Wednesday, November 07, 2012 7:36 AM
>>>> To: [hidden email]
>>>> Subject: Re: Why perform Methanol, Methanol/Acetone fixation at -20C
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> 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
>>>> <[hidden email]> wrote:
>>>>>
>>>>> *****
>>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>>> *****
>>>>>
>>>>> 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