aliquoting 4% formaldehyde

[Cromey, Douglas W - (dcromey)]

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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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

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[Marshak, David W]

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We make fresh formaldehyde from paraformaldehyde every time.  “10%” neutral buffered formalin keeps well at room temperature, but it contains methanol.

David W. Marshak, Ph.D., Professor

Department of Neurobiology and Anatomy

McGovern Medical School, UTHealth

6431 Fannin Street, Houston, TX 77030

phone: [phone]

From: "[hidden email]" <[hidden email]> on behalf of "Cromey, Douglas W - (dcromey)" <[hidden email]>
Reply-To: "[hidden email]" <[hidden email]>
Date: Tuesday, September 20, 2016 at 9:34 AM
To: "[hidden email]" <[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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Seamus Holden-2]

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At my old lab we regularly froze fresh aliquots of EM grade PFA at 16% concentration, -20C for single use in STORM/ PALM (eukaryotes and bacteria) and never had any problems.

 

Dr Seamus Holden

University Research Fellow

 

Centre for Bacterial Cell Biology

Baddiley-Clark Building

Newcastle University

Richardson Road

Newcastle upon Tyne

NE2 4AX, United Kingdom

 

Phone: [phone]

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
Sent: 20 September 2016 15:34
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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Raghavendra Palanakar]

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We always keep 4% PFA in PBS frozen as aliquots in minus 20 deg. C and never had a problem with fixation and never had issues with cell fixation artifacts.

 

And also, we use commercial  4% PFA stored at 4-8 deg. C, never had issues either.

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
Sent: Tuesday, September 20, 2016 4:34 PM
To: [hidden email]
Subject: [CONFOCALMICROSCOPY] 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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Martin Wessendorf-2]

***** 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. ***** Dear Doug--

My understanding is that if you cool solutions containing formaldehyde gas, you accelerate its re-conversion back to paraformaldehyde. 
However, I don't have a reference for this at my fingertips. 

--Did the solution freeze solid?  If so, the solubility of FA gas might've decreased when the phase changed and you might've lost some, but that's a guess. 

Good luck!

Martin Wessendorf

On 9/20/2016 9:34 AM, Cromey, Douglas W - (dcromey) wrote:

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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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Aleem]

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Dear Doug,

It is a common practice to aliquote 4%PFA immediately after preparation for later use. However, once thawed, leftover PFA shouldn't be reused.

Best,
Aleem

***** To join, leave or search the confocal microscopy listserv, go to:http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images onhttp://www.imgur.comand 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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463             email:[hidden email]

voice: <a href="tel:[phone]">520-626-2824      fax: <a href="tel:[phone]">520-626-2097

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance - http://microscopy.arizona.edu

 

[mmodel]

***** 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. *****

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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Michelle Peckham]

***** 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. *****

We also use frozen aliquots of frozen 4% paraformaldehyde, with no problems. Best to check how it was made – pH can be off for example -

 

From: Confocal Microscopy List <[hidden email]> on behalf of "MODEL, MICHAEL" <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Tuesday, 20 September 2016 at 15:47
To: "[hidden email]" <[hidden email]>
Subject: Re: 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. *****

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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Katrin Roth-2]

***** 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. ***** Hi Doug,

during my PhD I worked a lot with ampullae of  20% formaldehyde (Science Services) and diluted it to a 4% formaldehyde solution. It was used for fixation of mouse organs. The rest of the solution I always froze and it worked fine.

Katrin

––––––––––––––––––––––––––––––––
Dr. Katrin Roth
Centre of cancer and Immunology
Core Facility Cellular Imaging
Philipps University Marburg
Hans-Meerwein-Straße 3
35043 Marburg 
Germany

Am 20.09.2016 um 16:34 schrieb "Cromey, Douglas W - (dcromey)" <[hidden email]>:

***** 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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[mmodel]

***** 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. *****

That’s right, PFA it is usually prepared in PBS

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Michelle Peckham
Sent: Tuesday, September 20, 2016 10:50 AM
To: [hidden email]
Subject: Re: 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. *****

We also use frozen aliquots of frozen 4% paraformaldehyde, with no problems. Best to check how it was made – pH can be off for example -

 

From: Confocal Microscopy List <[hidden email]> on behalf of "MODEL, MICHAEL" <[hidden email]>
Reply-To: Confocal Microscopy List <[hidden email]>
Date: Tuesday, 20 September 2016 at 15:47
To: "[hidden email]" <[hidden email]>
Subject: Re: 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. *****

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 [[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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Alison J. North]

***** 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. *****

Hi Doug,

For EM I always made up fresh formaldehyde from PFA powder and used it immediately.  However, for light microscopy on tissue culture cells we typically use frozen aliquots stored at -20.  I guess the question partly depends on the level of resolution you are going to achieve - it's possible that something like STORM might reveal ultrastructural changes caused by using frozen vs. fresh formaldehyde, but on a regular microscope I've never noticed it.  Choosing the correct buffer is more of an issue, people can be very sloppy about that.

Best,

Alison

On 9/20/2016 10:34 AM, Cromey, Douglas W - (dcromey) wrote:

***** 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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Michal Opas]

***** 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. ***** Oh well, a sign of progress: he Googled it.
Now:
Formaldehyde: did he wonder why people routinely report 3.7% concentration as a fixative? 
"People" have been buying formaldehyde solution from a NNN company, which is 37-37% in water and dilute it 10x. Commercial product has methanol added to prevent polymerization. Store at room temperature.
People who wanted to invest work in having clean fix (formaldehyde soln has contaminants) did not freeze fix but invested work in preparing a fresh fixative (usually 4%) from parafomaldehyde powder for immediate use. No freezing again.
I hope this helps.
Michal

On 20/09/2016 10:34, Cromey, Douglas W - (dcromey) wrote:

***** 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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Sylvie Le Guyader]

***** 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. *****

From my experience of working with filopodia which retract/detach very easily:

Prepare 8% formaldehyde in PBS from powder (should be completely dissolved) and freeze it at -20 deg C in 15ml tubes (low volume/surface ratio). This is stable litterally for years. When needed, thaw an aliquot and keep it in the fridge for about 2 months. Warm up just the amount you need to 37 deg C, add it Vol/vol to your warm sample without shocking the cells by rinsing them with PBS. Incubate 5 min at 37 deg C (for a cell monolayer). then rinse with PBS.

It is easy to spot if cells in a monolayer suffer during fixation. Filopodia are not straight, cells detach easily during gentle washing, parts of the cell fold onto the cells. See an example here.

lately some of our users started using small capsules of pre made 4% PFA. Apart from the terrible waste in packaging, we have encountered a much higher than normal number of cases of folded cells.

Med vänlig hälsning / Best regards

Sylvie

@@@@@@@@@@@@@@@@@@@@@@@@

Sylvie Le Guyader, PhD

Live Cell Imaging Facility Manager

Karolinska Institutet- Bionut Dpt

Hälsovägen 7,

Novum, G lift, floor 6

14157 Huddinge

Sweden

mobile: [phone]

office: [phone] New number!

LCI website

---

From: Confocal Microscopy List [[hidden email]] on behalf of Cromey, Douglas W - (dcromey) [[hidden email]]
Sent: 20 September 2016 16:34
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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

[Mark A. Sanders]

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Hi Doug,

Like many others we freeze our freshly- made, buffered formaldehyde fix for light microscooy at -20C , but find it critcal to check and adjust the pH as needed after warming to the appropriate temperature.  Repeated freeze thaws are not a good thing so aliquoting to the amount that works for you routinely is a good idea.

Cheers,
Mark
*************************************
Mark A. Sanders      University of Minnesota
Program Director      Twin Cities Campus
University Imaging Centers  
www.uic.umn.edu

On Sep 20, 2016 9:34 AM, "Cromey, Douglas W - (dcromey)" <[hidden email]> wrote:

***** 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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  <a href="tel:[phone]" value="[phone]" target="_blank">520-626-2824       fax:  <a href="tel:[phone]" value="[phone]" target="_blank">520-626-2097

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

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[Jeffrey Carmichael]

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Hi Doug,

 

It probably depends on the level of resolution required which in turn determines the degree to which the available “fixable” structures in a cell must be tightly crosslinked.

 

I tend to be a bit on the OCD side when it comes to handling/preparing cells for microscopy.  I suspect that super resolution techniques will find evidence of slight differences in fixation that might not be noticed by looking at things like the morphology of filipodia, etc.

 

I can’t comment on precisely what the effects of less than optimal fixation look like, but I always made up fixative fresh from desiccated paraformaldehyde and would only use that same day. 

 

Formaldehyde solution begins to polymerize immediately.  Formaldehyde forms so-called crosslinks of methylene bridges by reacting with amines, purines and several other moieties.   These crosslinks become longer when formed by polymerized formaldehyde.  My understanding is that this results in a less than optimal crosslinking arrangement, where adjacent crosslinked molecules may be spanned by longer crosslinks than might otherwise be formed.  In other words, the various structures fixed in the cell are not as tightly “zipped-up” as they might otherwise be.

 

Much of the literature regarding formaldehyde is more relevant to and drawn from longstanding anatomical fixation techniques with very thick specimens (pathology), and doesn’t offer much help to high-resolution fixation of layers of cells or very thin tissue sections.  You can get away with less careful chemistry when it comes to preserving organs and large specimens which fix for days vs. high resolution microscopy of single layers of cells or thin tissue which fix in minutes.

 

Jeff

 

Jeff Carmichael

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an employee owned company

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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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

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[Mel Symeonides]

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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:

> *****
> 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.
> *****
>
> 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
>
> ------------------------------------------------------------------------------------------
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>
> office:  LSN 463              email: [hidden email]<mailto:[hidden email]>
> voice:  520-626-2824       fax:  [phone]
>
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"
>
> UA Microscopy Alliance -  http://microscopy.arizona.edu<http://microscopy.arizona.edu/>
>
>

--
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]

[Pippa Hawes]

***** 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. *****

Hi Doug

 

We regularly freeze aliquots of 4% para for fixation for confocal microscopy and I’ve never seen any artefacts that can be attributed to this.  We make the aliquots up using paraformaldehyde and PBS, separate into 10ml aliquots in Universals and freeze at -20.  Once an aliquot is thawed and used, the left over is thrown away, we do not re-freeze.  The colour should be completely clear, if there is a very slight yellow tinge then polymerisation has occurred and I wouldn’t use it. 

 

I suspect there is a buffer related issue, as others have said, rather than a problem caused by freezing. 

 

Oh, and we make sure to store our fixatives (para or glut) in a different freezer to our antibodies as there will be a small amount of gas produced which will fix any protein hanging around in your freezer (fridge too, of course).

 

Good luck sorting it out!

Pippa

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Cromey, Douglas W - (dcromey)
Sent: 20 September 2016 15:34
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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

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[Knecht, David]

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What did the student make up the 4% aliquots from?  If from pure 16% EM grade ampules, it is likely OK.  If from standard 37% formaldehyde solution, I would be suspicious.  We stopped using the 37% many years ago and never had problems since.  We usually break open a 16%  ampule and keep it in a brown bottle in the hood at room temp for weeks to months and then discard and open another.  Dave

Dr. David Knecht
Professor
Department of Molecular and Cell Biology
University of Connecticut
91 N. Eagleville Rd.
Storrs, CT 06269
860-486-2200

> On Sep 20, 2016, at 10:34 AM, Cromey, Douglas W - (dcromey) <[hidden email]> wrote:
>
> ***** 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
>  
> ------------------------------------------------------------------------------------------
> Douglas W. Cromey, M.S. - Associate Scientific Investigator
> Dept. of Cellular & Molecular Medicine, University of Arizona
> 1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA
>  
> office:  LSN 463              email: [hidden email]
> voice:  520-626-2824       fax:  [phone]
>  
> http://swehsc.pharmacy.arizona.edu/micro
> Home of: "Microscopy and Imaging Resources on the WWW"
>  
> UA Microscopy Alliance -  http://microscopy.arizona.edu

[Paul Rigby-2]

***** 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. *****

Hi All,

This is an interesting discussion that has relevance to all of our microscopy. I have a couple of comments:

1.       I have always used formaldehyde fixative freshly prepared from paraformaldehyde on the same day it was needed.

2.       When I was tempted to freeze aliquots to save time later on, upon thawing the aliquot I regularly saw small amounts of white precipitate on the wall of the container – the longer the storage, the worse the precipitate became. I interpreted this to be repolymerised formaldehyde (newly formed paraformaldehyde). While I have not checked, the chemistry of this repolymerisation process suggests that the pH of the solution should decrease – something that would not be good for fixation.

3.       If commercial formaldehyde solution (37% formaldehyde gas in water) needs to have methanol to prevent polymerisation, why do we think that freshly prepared formaldehyde (from paraformaldehyde) should be any different? Surely this suggest that fresh formaldehyde solution is unstable? Maybe freezing makes this even worse?

4.       In my older EM days, all of our fixatives were stored in a different fridge/freezer to where our antibodies were stored. As was explained to me at the time and as Pippa Hawes has pointed out, the activity of the antibodies could be affected by fixative vapours.

5.       As many people have already suggested, maybe, with super-resolution microscopy, artefacts from good or bad fixation will start to become more obvious.

Just my thoughts/recollections…

Paul Rigby

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Tuesday, 20 September 2016 10:43 PM
To: [hidden email]
Subject: Re: 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. *****

Dear Doug--

My understanding is that if you cool solutions containing formaldehyde gas, you accelerate its re-conversion back to paraformaldehyde. 
However, I don't have a reference for this at my fingertips. 

--Did the solution freeze solid?  If so, the solubility of FA gas might've decreased when the phase changed and you might've lost some, but that's a guess. 

Good luck!

Martin Wessendorf

On 9/20/2016 9:34 AM, Cromey, Douglas W - (dcromey) wrote:

***** 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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

-- 

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]

[Indig, Fred (NIH/NIA/IRP) [E]]

***** 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. *****

Dear Colleagues,

I have always used as stock Formaldehyde 10%, methanol free, Ultra Pure, from Polysciences (no commercial interest). Dilute before use, stored at room temp. Never noticed any fixation problems/artefacts, even under super res techniques like SIM and STED.

Fred E. Indig, Ph.D.

Head, Confocal Imaging Facility

Biomedical Research Center (BRC) Rm 8B135

National Institute on Aging/NIH

251 Bayview Blvd.

Baltimore, MD  21224-6825

Tel. [phone]

Fax  [phone]

[hidden email] 

 

---

From: Paul Rigby [[hidden email]]
Sent: Tuesday, September 20, 2016 11:55 PM
To: [hidden email]
Subject: Re: 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. *****

Hi All,

This is an interesting discussion that has relevance to all of our microscopy. I have a couple of comments:

1.       I have always used formaldehyde fixative freshly prepared from paraformaldehyde on the same day it was needed.

2.       When I was tempted to freeze aliquots to save time later on, upon thawing the aliquot I regularly saw small amounts of white precipitate on the wall of the container – the longer the storage, the worse the precipitate became. I interpreted this to be repolymerised formaldehyde (newly formed paraformaldehyde). While I have not checked, the chemistry of this repolymerisation process suggests that the pH of the solution should decrease – something that would not be good for fixation.

3.       If commercial formaldehyde solution (37% formaldehyde gas in water) needs to have methanol to prevent polymerisation, why do we think that freshly prepared formaldehyde (from paraformaldehyde) should be any different? Surely this suggest that fresh formaldehyde solution is unstable? Maybe freezing makes this even worse?

4.       In my older EM days, all of our fixatives were stored in a different fridge/freezer to where our antibodies were stored. As was explained to me at the time and as Pippa Hawes has pointed out, the activity of the antibodies could be affected by fixative vapours.

5.       As many people have already suggested, maybe, with super-resolution microscopy, artefacts from good or bad fixation will start to become more obvious.

Just my thoughts/recollections…

Paul Rigby

 

 

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Martin Wessendorf
Sent: Tuesday, 20 September 2016 10:43 PM
To: [hidden email]
Subject: Re: 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. *****

Dear Doug--

My understanding is that if you cool solutions containing formaldehyde gas, you accelerate its re-conversion back to paraformaldehyde. 
However, I don't have a reference for this at my fingertips. 

--Did the solution freeze solid?  If so, the solubility of FA gas might've decreased when the phase changed and you might've lost some, but that's a guess. 

Good luck!

Martin Wessendorf

On 9/20/2016 9:34 AM, Cromey, Douglas W - (dcromey) wrote:

***** 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

 

------------------------------------------------------------------------------------------

Douglas W. Cromey, M.S. - Associate Scientific Investigator

Dept. of Cellular & Molecular Medicine, University of Arizona

1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

 

office:  LSN 463              email: [hidden email]

voice:  520-626-2824       fax:  [phone]

 

http://swehsc.pharmacy.arizona.edu/micro

Home of: "Microscopy and Imaging Resources on the WWW"

 

UA Microscopy Alliance -  http://microscopy.arizona.edu

 

-- 

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]