Glyoxal versus PFA for immunofluorescence

[kspencer007]

*****
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;
                Have you tested glyoxal versus paraformaldehyde for immunostaining? Do you have a preference?  Do you need to choose your fixative depending upon your target? Curious if this is a good, safer replacement for routine PFA fixation.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035/

Best;
                Kathy Spencer

The Scripps Research Institute
Dept of Molecular and Cellular Neuroscience
10550 N. Torrey Pines Road
DNC 216
La Jolla, Ca 92037

[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.
*****

It’s worth a go, as it can be better, depending on your application/antibody. In one case for us, it has made a real difference- staining much improved.

You might want to try deionising the Glyoxal first.

All best

Michelle


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Kathryn Spencer <[hidden email]>
Sent: Thursday, October 18, 2018 10:30 pm
To: [hidden email]
Subject: Glyoxal versus PFA for immunofluorescence

*****
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;
Have you tested glyoxal versus paraformaldehyde for immunostaining? Do you have a preference? Do you need to choose your fixative depending upon your target? Curious if this is a good, safer replacement for routine PFA fixation.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035/

Best;
Kathy Spencer

The Scripps Research Institute
Dept of Molecular and Cellular Neuroscience
10550 N. Torrey Pines Road
DNC 216
La Jolla, Ca 92037

[Elisa D'Este]

*****
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 Kathy,

it depends on your target. In some cases the quality of the staining in
improved, in other cases the labeling is lost. Therefore, I would not
recommend switching completely to Glyoxal but decide depending on your
experiment.

Good luck!

Elisa

--
Elisa D’Este, Dr.
Head of the Light Microscopy Facility
Max-Planck-Institute for Medical Research
Jahnstraße 29
D-69120 Heidelberg
Phone: [phone]


On 18.10.2018 23:29, Kathryn Spencer 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 all;
>                  Have you tested glyoxal versus paraformaldehyde for immunostaining? Do you have a preference?  Do you need to choose your fixative depending upon your target? Curious if this is a good, safer replacement for routine PFA fixation.
>
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035/
>
> Best;
>                  Kathy Spencer
>
> The Scripps Research Institute
> Dept of Molecular and Cellular Neuroscience
> 10550 N. Torrey Pines Road
> DNC 216
> La Jolla, Ca 92037

[Valeria Berno-4]

*****
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 Michelle, may I ask you what do you mean for "deionising" the
Glyoaxial? and how do you perform this?

thanks

VAleria


Il 19/10/18 08:50, Michelle Peckham ha scritto:

> *****
> 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.
> *****
>
> It’s worth a go, as it can be better, depending on your application/antibody. In one case for us, it has made a real difference- staining much improved.
>
> You might want to try deionising the Glyoxal first.
>
> All best
>
> Michelle
>
>
> ________________________________
> From: Confocal Microscopy List <[hidden email]> on behalf of Kathryn Spencer <[hidden email]>
> Sent: Thursday, October 18, 2018 10:30 pm
> To: [hidden email]
> Subject: Glyoxal versus PFA for immunofluorescence
>
> *****
> 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;
> Have you tested glyoxal versus paraformaldehyde for immunostaining? Do you have a preference? Do you need to choose your fixative depending upon your target? Curious if this is a good, safer replacement for routine PFA fixation.
>
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035/
>
> Best;
> Kathy Spencer
>
> The Scripps Research Institute
> Dept of Molecular and Cellular Neuroscience
> 10550 N. Torrey Pines Road
> DNC 216
> La Jolla, Ca 92037

--
ALEMBIC
Advanced Light and Electron Microscopy BioImaging Center
San Raffaele Scientific Institute
DIBIT 1
via Olgettina 58, 20132 - Milano - Italy

Tel [phone]
Fax [phone]
e-mail: [hidden email]
home page:
http://alembic.hsr.it



Rispetta l’ambiente: non stampare questa mail se non è necessario.
Respect the environment: print this email only if necessary.

[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.
*****

Dear Valeria

There are quite a few protocols out there - so you can just 'google' it (or see http://cshprotocols.cshlp.org/content/2015/2/pdb.rec086538.short).  It's a method of removing impurities that might affect performance from the stock solution that one buys from Sigma.  In our hands this did help improve our results (though I admit it was someone else who did the de-ionisation). Glyoxal has been used a lot for RNA, and deionization is common for that application.

All best

Michelle

On 19/10/2018, 09:23, "Confocal Microscopy List on behalf of Berno Valeria" <[hidden email] on behalf of [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.
    *****
   
    Hi Michelle, may I ask you what do you mean for "deionising" the
    Glyoaxial? and how do you perform this?
   
    thanks
   
    VAleria
   
   
    Il 19/10/18 08:50, Michelle Peckham ha scritto:
    > *****
    > 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.
    > *****
    >
    > It’s worth a go, as it can be better, depending on your application/antibody. In one case for us, it has made a real difference- staining much improved.
    >
    > You might want to try deionising the Glyoxal first.
    >
    > All best
    >
    > Michelle
    >
    >
    > ________________________________
    > From: Confocal Microscopy List <[hidden email]> on behalf of Kathryn Spencer <[hidden email]>
    > Sent: Thursday, October 18, 2018 10:30 pm
    > To: [hidden email]
    > Subject: Glyoxal versus PFA for immunofluorescence
    >
    > *****
    > 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;
    > Have you tested glyoxal versus paraformaldehyde for immunostaining? Do you have a preference? Do you need to choose your fixative depending upon your target? Curious if this is a good, safer replacement for routine PFA fixation.
    >
    > https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035/
    >
    > Best;
    > Kathy Spencer
    >
    > The Scripps Research Institute
    > Dept of Molecular and Cellular Neuroscience
    > 10550 N. Torrey Pines Road
    > DNC 216
    > La Jolla, Ca 92037
   
    --
    ALEMBIC
    Advanced Light and Electron Microscopy BioImaging Center
    San Raffaele Scientific Institute
    DIBIT 1
    via Olgettina 58, 20132 - Milano - Italy
   
    Tel [phone]
    Fax [phone]
    e-mail: [hidden email]
    home page:
    http://alembic.hsr.it
   
   
   
    Rispetta l’ambiente: non stampare questa mail se non è necessario.
    Respect the environment: print this email only if necessary.
   

[Christian Soeller]

*****
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 Michelle,

It would be useful if somebody could study the effect of deionisation systematically for fixation. It is at least not obvious why this treatment (which is clearly well established for RNA preservation, a very labile molecule) is required for tissue/cell fixation. Anecdotal evidence can at times be highly misleading with fixation (as results can be variable at the best of times) and it would be good to know if the effort is indeed required and demonstrably helpful.

Not questioning what you say, just wondering how strong the evidence is out there. I guess the original paper did not investigate this?

Best,
Christian

> On 19/10/2018, at 10:49 am, Michelle Peckham <[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.
> *****
>
> Dear Valeria
>
> There are quite a few protocols out there - so you can just 'google' it (or see http://cshprotocols.cshlp.org/content/2015/2/pdb.rec086538.short).  It's a method of removing impurities that might affect performance from the stock solution that one buys from Sigma.  In our hands this did help improve our results (though I admit it was someone else who did the de-ionisation). Glyoxal has been used a lot for RNA, and deionization is common for that application.
>
> All best
>
> Michelle
>
> On 19/10/2018, 09:23, "Confocal Microscopy List on behalf of Berno Valeria" <[hidden email] on behalf of [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.
>    *****
>
>    Hi Michelle, may I ask you what do you mean for "deionising" the
>    Glyoaxial? and how do you perform this?
>
>    thanks
>
>    VAleria
>
>
>    Il 19/10/18 08:50, Michelle Peckham ha scritto:
>> *****
>> 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.
>> *****
>>
>> It’s worth a go, as it can be better, depending on your application/antibody. In one case for us, it has made a real difference- staining much improved.
>>
>> You might want to try deionising the Glyoxal first.
>>
>> All best
>>
>> Michelle
>>
>>
>> ________________________________
>> From: Confocal Microscopy List <[hidden email]> on behalf of Kathryn Spencer <[hidden email]>
>> Sent: Thursday, October 18, 2018 10:30 pm
>> To: [hidden email]
>> Subject: Glyoxal versus PFA for immunofluorescence
>>
>> *****
>> 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;
>> Have you tested glyoxal versus paraformaldehyde for immunostaining? Do you have a preference? Do you need to choose your fixative depending upon your target? Curious if this is a good, safer replacement for routine PFA fixation.
>>
>> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035/
>>
>> Best;
>> Kathy Spencer
>>
>> The Scripps Research Institute
>> Dept of Molecular and Cellular Neuroscience
>> 10550 N. Torrey Pines Road
>> DNC 216
>> La Jolla, Ca 92037
>
>    --
>    ALEMBIC
>    Advanced Light and Electron Microscopy BioImaging Center
>    San Raffaele Scientific Institute
>    DIBIT 1
>    via Olgettina 58, 20132 - Milano - Italy
>
>    Tel [phone]
>    Fax [phone]
>    e-mail: [hidden email]
>    home page:
>    http://alembic.hsr.it
>
>
>
>    Rispetta l’ambiente: non stampare questa mail se non è necessario.
>    Respect the environment: print this email only if necessary.
>
>

[Dun, Alison]

*****
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 Valeria,
Silvio Rizzoli has worked a lot on fixation and the use of Glyoxal, he is very helpful so you could always drop him an email,
Good luck!
Ali



On 19 Oct 2018, at 10:49, Michelle Peckham <[hidden email]<mailto:[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.
*****

Dear Valeria

There are quite a few protocols out there - so you can just 'google' it (or see http://cshprotocols.cshlp.org/content/2015/2/pdb.rec086538.short).  It's a method of removing impurities that might affect performance from the stock solution that one buys from Sigma.  In our hands this did help improve our results (though I admit it was someone else who did the de-ionisation). Glyoxal has been used a lot for RNA, and deionization is common for that application.

All best

Michelle

On 19/10/2018, 09:23, "Confocal Microscopy List on behalf of Berno Valeria" <[hidden email] on behalf of [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.
   *****

   Hi Michelle, may I ask you what do you mean for "deionising" the
   Glyoaxial? and how do you perform this?

   thanks

   VAleria


   Il 19/10/18 08:50, Michelle Peckham ha scritto:
*****
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.
*****

It’s worth a go, as it can be better, depending on your application/antibody. In one case for us, it has made a real difference- staining much improved.

You might want to try deionising the Glyoxal first.

All best

Michelle


________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Kathryn Spencer <[hidden email]>
Sent: Thursday, October 18, 2018 10:30 pm
To: [hidden email]
Subject: Glyoxal versus PFA for immunofluorescence

*****
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;
Have you tested glyoxal versus paraformaldehyde for immunostaining? Do you have a preference? Do you need to choose your fixative depending upon your target? Curious if this is a good, safer replacement for routine PFA fixation.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035/

Best;
Kathy Spencer

The Scripps Research Institute
Dept of Molecular and Cellular Neuroscience
10550 N. Torrey Pines Road
DNC 216
La Jolla, Ca 92037

   --
   ALEMBIC
   Advanced Light and Electron Microscopy BioImaging Center
   San Raffaele Scientific Institute
   DIBIT 1
   via Olgettina 58, 20132 - Milano - Italy

   Tel [phone]
   Fax [phone]
   e-mail: [hidden email]
   home page:
   http://alembic.hsr.it



   Rispetta l’ambiente: non stampare questa mail se non è necessario.
   Respect the environment: print this email only if necessary.



________________________________

Heriot-Watt University is The Times & The Sunday Times International University of the Year 2018

Founded in 1821, Heriot-Watt is a leader in ideas and solutions. With campuses and students across the entire globe we span the world, delivering innovation and educational excellence in business, engineering, design and the physical, social and life sciences.

This email is generated from the Heriot-Watt University Group, which includes:

  1.  Heriot-Watt University, a Scottish charity registered under number SC000278
  2.  Edinburgh Business School a Charity Registered in Scotland, SC026900. Edinburgh Business School is a company limited by guarantee, registered in Scotland with registered number SC173556 and registered office at Heriot-Watt University Finance Office, Riccarton, Currie, Midlothian, EH14 4AS
  3.  Heriot- Watt Services Limited (Oriam), Scotland's national performance centre for sport. Heriot-Watt Services Limited is a private limited company registered is Scotland with registered number SC271030 and registered office at Research & Enterprise Services Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS.

The contents (including any attachments) are confidential. If you are not the intended recipient of this e-mail, any disclosure, copying, distribution or use of its contents is strictly prohibited, and you should please notify the sender immediately and then delete it (including any attachments) from your system.

[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.
*****

Not in the original paper - but we've tried both, not done an extensive characterisation, but the deionised stuff seems to work better.
We'll try to get some better direct comparisons done.

M

On 19/10/2018, 11:29, "Confocal Microscopy List on behalf of Christian Soeller" <[hidden email] on behalf of [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.
    *****
   
    Dear Michelle,
   
    It would be useful if somebody could study the effect of deionisation systematically for fixation. It is at least not obvious why this treatment (which is clearly well established for RNA preservation, a very labile molecule) is required for tissue/cell fixation. Anecdotal evidence can at times be highly misleading with fixation (as results can be variable at the best of times) and it would be good to know if the effort is indeed required and demonstrably helpful.
   
    Not questioning what you say, just wondering how strong the evidence is out there. I guess the original paper did not investigate this?
   
    Best,
    Christian
   
   
    > On 19/10/2018, at 10:49 am, Michelle Peckham <[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.
    > *****
    >
    > Dear Valeria
    >
    > There are quite a few protocols out there - so you can just 'google' it (or see http://cshprotocols.cshlp.org/content/2015/2/pdb.rec086538.short).  It's a method of removing impurities that might affect performance from the stock solution that one buys from Sigma.  In our hands this did help improve our results (though I admit it was someone else who did the de-ionisation). Glyoxal has been used a lot for RNA, and deionization is common for that application.
    >
    > All best
    >
    > Michelle
    >
    > On 19/10/2018, 09:23, "Confocal Microscopy List on behalf of Berno Valeria" <[hidden email] on behalf of [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.
    >    *****
    >
    >    Hi Michelle, may I ask you what do you mean for "deionising" the
    >    Glyoaxial? and how do you perform this?
    >
    >    thanks
    >
    >    VAleria
    >
    >
    >    Il 19/10/18 08:50, Michelle Peckham ha scritto:
    >> *****
    >> 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.
    >> *****
    >>
    >> It’s worth a go, as it can be better, depending on your application/antibody. In one case for us, it has made a real difference- staining much improved.
    >>
    >> You might want to try deionising the Glyoxal first.
    >>
    >> All best
    >>
    >> Michelle
    >>
    >>
    >> ________________________________
    >> From: Confocal Microscopy List <[hidden email]> on behalf of Kathryn Spencer <[hidden email]>
    >> Sent: Thursday, October 18, 2018 10:30 pm
    >> To: [hidden email]
    >> Subject: Glyoxal versus PFA for immunofluorescence
    >>
    >> *****
    >> 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;
    >> Have you tested glyoxal versus paraformaldehyde for immunostaining? Do you have a preference? Do you need to choose your fixative depending upon your target? Curious if this is a good, safer replacement for routine PFA fixation.
    >>
    >> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5753035/
    >>
    >> Best;
    >> Kathy Spencer
    >>
    >> The Scripps Research Institute
    >> Dept of Molecular and Cellular Neuroscience
    >> 10550 N. Torrey Pines Road
    >> DNC 216
    >> La Jolla, Ca 92037
    >
    >    --
    >    ALEMBIC
    >    Advanced Light and Electron Microscopy BioImaging Center
    >    San Raffaele Scientific Institute
    >    DIBIT 1
    >    via Olgettina 58, 20132 - Milano - Italy
    >
    >    Tel [phone]
    >    Fax [phone]
    >    e-mail: [hidden email]
    >    home page:
    >    http://alembic.hsr.it
    >
    >
    >
    >    Rispetta l’ambiente: non stampare questa mail se non è necessario.
    >    Respect the environment: print this email only if necessary.
    >
    >