Paraformaldehyde and permeabilization

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Trop, Stefanie A. Trop, Stefanie A.
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Paraformaldehyde and permeabilization

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Dear list,

Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!

Yours,
Stefanie Trop

_____________________________________________
Stefanie Trop, PhD Candidate
Johns Hopkins Bloomberg School of Public Health
Department of Molecular Microbiology and Immunology
Laboratory of Jelena Levitskaya

[hidden email]
(410) 502-9134
Michelle Peckham Michelle Peckham
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Re: Paraformaldehyde and permeabilization

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Yes, you do get partial permeabilisation with para formaldehyde.
You can stain unfixed non permeabilised cells to look at membrane proteins.

Michelle

On 21 Aug 2012, at 20:38, "Trop, Stefanie A." <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!
>
> Yours,
> Stefanie Trop
>
> _____________________________________________
> Stefanie Trop, PhD Candidate
> Johns Hopkins Bloomberg School of Public Health
> Department of Molecular Microbiology and Immunology
> Laboratory of Jelena Levitskaya
>
> [hidden email]
> (410) 502-9134
Julia Edgar Julia Edgar
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Re: Paraformaldehyde and permeabilization

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I agree. Live cell staining (without fixation or permeabilisation) circumvents this problem.
Julia
 
 
Yes, you do get partial permeabilisation with para formaldehyde.
You can stain unfixed non permeabilised cells to look at membrane proteins.

Michelle

On 21 Aug 2012, at 20:38, "Trop, Stefanie A." <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!
>
> Yours,
> Stefanie Trop
>
> _____________________________________________
> Stefanie Trop, PhD Candidate
> Johns Hopkins Bloomberg School of Public Health
> Department of Molecular Microbiology and Immunology
> Laboratory of Jelena Levitskaya
>
> [hidden email]
> (410) 502-9134
mmodel mmodel
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Re: Paraformaldehyde and permeabilization

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Formaldehyde normally makes membranes permeable only to some small molecules, not to antibodies.  

Mike Model


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Trop, Stefanie A.
Sent: Tuesday, August 21, 2012 3:26 PM
To: [hidden email]
Subject: Paraformaldehyde and permeabilization

*****
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Dear list,

Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!

Yours,
Stefanie Trop

_____________________________________________
Stefanie Trop, PhD Candidate
Johns Hopkins Bloomberg School of Public Health
Department of Molecular Microbiology and Immunology
Laboratory of Jelena Levitskaya

[hidden email]
(410) 502-9134
Masur, Sandra Masur, Sandra
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Re: Paraformaldehyde and permeabilization

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Live cell staining should be done on ice if possible to prevent endocytosis of the antibody during the staining. Fixation should follow staining and then if necessary the application of a secondary antibody.
Best regards,

Sandy
__________________________
Sandra K. Masur, PhD
Professor, Ophthalmology
Mount Sinai School of Medicine, Box 1183
1 Gustave Levy Place
New York NY 10029-6574

[hidden email]<mailto:[hidden email]>
telephone: 212-241-0089
fax: 212-289-5945



On Aug 21, 2012, at 3:55 PM, Julia Edgar wrote:

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I agree. Live cell staining (without fixation or permeabilisation) circumvents this problem.
Julia


Yes, you do get partial permeabilisation with para formaldehyde.
You can stain unfixed non permeabilised cells to look at membrane proteins.

Michelle

On 21 Aug 2012, at 20:38, "Trop, Stefanie A." <[hidden email]> wrote:

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Dear list,

Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!

Yours,
Stefanie Trop

_____________________________________________
Stefanie Trop, PhD Candidate
Johns Hopkins Bloomberg School of Public Health
Department of Molecular Microbiology and Immunology
Laboratory of Jelena Levitskaya

[hidden email]
(410) 502-9134
Christian Soeller Christian Soeller
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Re: Paraformaldehyde and permeabilization

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Some years ago we tried staining an intracellular protein without permeabilization, just two % pfa for 10 minutes. We got beautiful staining, indistinguishable from that in explicitly permeabilized cells. So antibodies can clearly get through and we have repeatedly verified that since then. Live cell surface labelling seems the way to go but requires an extracellular epitope.
Christian
--
Sent from my Android phone with K-9 Mail. Please excuse my brevity.

"MODEL, MICHAEL" <[hidden email]> wrote:

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Formaldehyde normally makes membranes permeable only to some small molecules, not to antibodies.

Mike Model


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Trop, Stefanie A.
Sent: Tuesday, August 21, 2012 3:26 PM
To: [hidden email]
Subject: Paraformaldehyde and permeabilization

*****
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Dear list,

Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!

Yours,
Stefanie Trop

_____________________________________________

Stefanie Trop, PhD Candidate
Johns Hopkins Bloomberg School of Public Health
Department of Molecular Microbiology and Immunology
Laboratory of Jelena Levitskaya

[hidden email]
(410) 502-9134
Tim Feinstein-2 Tim Feinstein-2
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Re: Paraformaldehyde and permeabilization

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Yep, in my lab's experience you cannot have PFA fixation without permeabilization.  In some cases  an additional permeabilizing step improves staining, but some intracellular staining is inevitable after PFA.  

However, Ms. Trop's problem is the reverse.  I would not panic a priori if I saw ubiquitinated proteins on the plasma membrane, but as to why you might find EEA1 on the plasma membrane, you have a few options.  Your labmate may have overfixed her cells, made a poor batch of staining buffer or in some other way interfered with the antibody's specificity.  You may have a batch of antibody that is old, not very good or which targets the wrong species.  Finally, an event that causes a large amount of PI3P to appear on the plasma membrane, for example lots of simultaneous clathrin-mediated endocytosis, could cause EEA1 to localize there temporarily.  In fact the many things that might cause EEA1 to go to the PM would probably involve an increase in ubiquitinated proteins on the PM as well.  

To sort these possibilities out, try starting with a western blot.  If you get a clean band from this cell line at about 150-160 kDa, re-make the IF buffer solutions and try again.  Include cells with different treatment conditions (e.g., serum-starved for a while) to rule out the possibility that this is a weird positive result.  

cheers,


TF

Timothy Feinstein, PhD
Postdoctoral Fellow
Laboratory for GPCR Biology
Dept. of Pharmacology & Chemical Biology
University of Pittsburgh, School of Medicine
BST W1301, 200 Lothrop St.
Pittsburgh, PA  15261

On Aug 21, 2012, at 4:45 PM, Christian Soeller wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Some years ago we tried staining an intracellular protein without permeabilization, just two % pfa for 10 minutes. We got beautiful staining, indistinguishable from that in explicitly permeabilized cells. So antibodies can clearly get through and we have repeatedly verified that since then. Live cell surface labelling seems the way to go but requires an extracellular epitope.
> Christian
> --
> Sent from my Android phone with K-9 Mail. Please excuse my brevity.
>
> "MODEL, MICHAEL" <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Formaldehyde normally makes membranes permeable only to some small molecules, not to antibodies.
>
> Mike Model
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Trop, Stefanie A.
> Sent: Tuesday, August 21, 2012 3:26 PM
> To: [hidden email]
> Subject: Paraformaldehyde and permeabilization
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!
>
> Yours,
> Stefanie Trop
>
> _____________________________________________
>
> Stefanie Trop, PhD Candidate
> Johns Hopkins Bloomberg School of Public Health
> Department of Molecular Microbiology and Immunology
> Laboratory of Jelena Levitskaya
>
> [hidden email]
> (410) 502-9134
Paul Rigby-2 Paul Rigby-2
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Re: Paraformaldehyde and permeabilization

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Hi All,

We get asked these questions all the time and I can say, without doubt, that paraformaldehyde fixation will permeabilise cell membranes. Whether paraformaldehyde fixation alone will give your antibodies access to your antigen epitope is a totally different question and permeabilisation may additionally be required. This is likely why coagulative fixatives (like methanol and acetone) work well in some situations since they likely cause both fixation and permeabilisation at the same time. Similarly, antigen retrieval (perhaps also able to be called protein tertiary structure disruption) may also be required to sterically allow antibody molecules access to the antigen epitope. Exactly what is required to get good antibody staining is multi-factorial and is definitely dependent upon the particular antibody antigen epitope combination.

Beware, there is a massive amount of misinformation (or missing information) in the literature about antibody staining. Read widely, do appropriate testing and use suitable controls before publishing.

Sorry, I'll get off my soapbox now.
Cheers all
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 Trop, Stefanie A.
Sent: Wednesday, 22 August 2012 3:26 AM
To: [hidden email]
Subject: Paraformaldehyde and permeabilization

*****
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Dear list,

Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!

Yours,
Stefanie Trop

_____________________________________________
Stefanie Trop, PhD Candidate
Johns Hopkins Bloomberg School of Public Health Department of Molecular Microbiology and Immunology Laboratory of Jelena Levitskaya

[hidden email]
(410) 502-9134
Michael Abanto Michael Abanto
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Re: Paraformaldehyde and permeabilization

In reply to this post by Julia Edgar
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Hi everybody,

I would also surface label the cells by cooling them to 4C - to block endocytosis. PFA definitely permeabilizes the PM to some degree in my experience, and it sounds like this is the case for you.
Another factor you need to consider is that some 'intracellular' organelles (including endosomes in some cell types) are surface accessible. these surface accessible compartments can be distinguished by live staining at 4C and quenching/stripping/ blocking etc.
Finally, whatever you end up doing, you can show that you labeled the surface by washing, fixing, and then staining after permeabilization.

Mike







On Aug 21, 2012, at 9:55 PM, Julia Edgar wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I agree. Live cell staining (without fixation or permeabilisation) circumvents this problem.
> Julia
>
>
> Yes, you do get partial permeabilisation with para formaldehyde.
> You can stain unfixed non permeabilised cells to look at membrane proteins.
>
> Michelle
>
> On 21 Aug 2012, at 20:38, "Trop, Stefanie A." <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Dear list,
>>
>> Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!
>>
>> Yours,
>> Stefanie Trop
>>
>> _____________________________________________
>> Stefanie Trop, PhD Candidate
>> Johns Hopkins Bloomberg School of Public Health
>> Department of Molecular Microbiology and Immunology
>> Laboratory of Jelena Levitskaya
>>
>> [hidden email]
>> (410) 502-9134
Rosemary.White Rosemary.White
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Re: Paraformaldehyde and permeabilization

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Dear all,

In plant immumo work, we routinely use just paraformaldehyde fixation before immunolabelling of fresh hand sections, and only resort to other permeabilising agents if absolutely necessary.

Cheers,
Rosemary

Dr Rosemary White
CSIRO Plant Industry
GPO Box 1600
Canberra, ACT 2601
Australia

T 61 2 6246 5475
F 61 2 6246 5334
________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Paul Rigby [[hidden email]]
Sent: Wednesday, 22 August 2012 10:09 a.m.
To: [hidden email]
Subject: Re: Paraformaldehyde and permeabilization

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Hi All,

We get asked these questions all the time and I can say, without doubt, that paraformaldehyde fixation will permeabilise cell membranes. Whether paraformaldehyde fixation alone will give your antibodies access to your antigen epitope is a totally different question and permeabilisation may additionally be required. This is likely why coagulative fixatives (like methanol and acetone) work well in some situations since they likely cause both fixation and permeabilisation at the same time. Similarly, antigen retrieval (perhaps also able to be called protein tertiary structure disruption) may also be required to sterically allow antibody molecules access to the antigen epitope. Exactly what is required to get good antibody staining is multi-factorial and is definitely dependent upon the particular antibody antigen epitope combination.

Beware, there is a massive amount of misinformation (or missing information) in the literature about antibody staining. Read widely, do appropriate testing and use suitable controls before publishing.

Sorry, I'll get off my soapbox now.
Cheers all
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 Trop, Stefanie A.
Sent: Wednesday, 22 August 2012 3:26 AM
To: [hidden email]
Subject: Paraformaldehyde and permeabilization

*****
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Dear list,

Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!

Yours,
Stefanie Trop

_____________________________________________
Stefanie Trop, PhD Candidate
Johns Hopkins Bloomberg School of Public Health Department of Molecular Microbiology and Immunology Laboratory of Jelena Levitskaya

[hidden email]
(410) 502-9134
mmodel mmodel
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Re: Paraformaldehyde and permeabilization

In reply to this post by Paul Rigby-2
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I guess I was wrong. But what we have certainly checked is that permeabilization by PFA does not happen until the fixative is removed (Pelts et al, Biotechniques, 2011). Of course you add antibodies only after a wash... -

Mike

________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Paul Rigby [[hidden email]]
Sent: Tuesday, August 21, 2012 8:09 PM
To: [hidden email]
Subject: Re: Paraformaldehyde and permeabilization

*****
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Hi All,

We get asked these questions all the time and I can say, without doubt, that paraformaldehyde fixation will permeabilise cell membranes. Whether paraformaldehyde fixation alone will give your antibodies access to your antigen epitope is a totally different question and permeabilisation may additionally be required. This is likely why coagulative fixatives (like methanol and acetone) work well in some situations since they likely cause both fixation and permeabilisation at the same time. Similarly, antigen retrieval (perhaps also able to be called protein tertiary structure disruption) may also be required to sterically allow antibody molecules access to the antigen epitope. Exactly what is required to get good antibody staining is multi-factorial and is definitely dependent upon the particular antibody antigen epitope combination.

Beware, there is a massive amount of misinformation (or missing information) in the literature about antibody staining. Read widely, do appropriate testing and use suitable controls before publishing.

Sorry, I'll get off my soapbox now.
Cheers all
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 Trop, Stefanie A.
Sent: Wednesday, 22 August 2012 3:26 AM
To: [hidden email]
Subject: Paraformaldehyde and permeabilization

*****
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Dear list,

Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!

Yours,
Stefanie Trop

_____________________________________________
Stefanie Trop, PhD Candidate
Johns Hopkins Bloomberg School of Public Health Department of Molecular Microbiology and Immunology Laboratory of Jelena Levitskaya

[hidden email]
(410) 502-9134
Francisco J H Blazquez Francisco J H Blazquez
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Re: Paraformaldehyde and permeabilization

In reply to this post by Trop, Stefanie A.
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Part of problem may be due to the solubility of the antigens and the
fixation process. When PFA is added to the cells, if the antigen is not
firmly attached to the cytoskeleton or inside some organelle, it may be
displaced by the fluids movements inside the cell when the slide is
washed. The fluids leak out the cell but the soluble antigen is retained
by the cell membrane. It is a well known phenomenon in tissue fixation,
specially if we want to observe glycogen when this "wave" effect is
easlly seen. One solution may be to apply the fixative and the washing
at low temperature, as low as possible, to slow the movement of fluids
inside the cytoplasm. Or to use acetone (a coagulative fixative) at -20
celsius degrees.

Em 21/08/2012 16:25, Trop, Stefanie A. escreveu:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Dear list,
>
> Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!
>
> Yours,
> Stefanie Trop
>
> _____________________________________________
> Stefanie Trop, PhD Candidate
> Johns Hopkins Bloomberg School of Public Health
> Department of Molecular Microbiology and Immunology
> Laboratory of Jelena Levitskaya
>
> [hidden email]
> (410) 502-9134
>
> Prof. Dr. Francisco Javier Hernandez Blazquez
> University of São Paulo
> School of Veterinary Medicine and Animal Science
> Departament of Surgery - Sector of Anatomy
> Av. Prof. Dr. Orlando Marques de Paiva, 87
> 05508-270 - São Paulo (SP) - Brazil
> http:/mav.fmvz.usp.br
> Tel..55 (11) 3091 1374  Fax  55 (11) 3091 7805
> email: [hidden email]
>
Gert van Cappellen-2 Gert van Cappellen-2
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Re: Paraformaldehyde and permeabilization

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We did some test with 4% PFA on a microscope and studied the penetration
of propidium iodide (PI) into the cell and nucleus in GFP transfected
cells. The moment you add the PFA the GFP signal drops to about 50%. The
nucleus does not become red till 2 to 3h after the PFS fixation. Than
one cell after the other started to get a red nucleus and the GFP signal
came back to 80% of the original signal.   Probably long fixation will
increase the permeability of the membranes.

Best regards,
Gert

Gert van Cappellen
Optical Imaging Centre
Erasmus MC (Erasmus Medical Centre)
Rotterdam
Netherlands


On 21/08/2012 21:25, Trop, Stefanie A. wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
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> *****
>
> Dear list,
>
> Has anyone had experience with paraformaldehyde permeabilizing mammalian cells? One of my labmates fixed cells grown on coverslips with a fresh aliquot of 4% paraformaldehyde that we made from powder and had stored at -20C, and had strong staining with antibodies that I would not expect to be present on the membrane, specifically EEA1 and ubiquitin-conjugated proteins. He did not add any alcohols nor detergents during any subsequent staining steps. Thanks for any assistance you can offer!
>
> Yours,
> Stefanie Trop
>
> _____________________________________________
> Stefanie Trop, PhD Candidate
> Johns Hopkins Bloomberg School of Public Health
> Department of Molecular Microbiology and Immunology
> Laboratory of Jelena Levitskaya
>
> [hidden email]
> (410) 502-9134
Teng-Leong Chew Teng-Leong Chew
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Staining 3D collagen gel

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Hi all,

Can anyone recommend a collagen staining protocol that is
compatible with fluorescence live cell imaging in 3-D?

We are currently doing cell migration assay and would like
to mark the demarcation of the collagen gel (which we make
from rat tails) and the culture medium very clearly. In
addition, it would be nice to see the tunnels as cells
go through the gel as well.

Thanks!!

Regards,
Leong

--
Teng-Leong Chew, PhD

Director, Cell Imaging Facility & Nikon Imaging Center
Northwestern University
312-503-2841
mcammer mcammer
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Re: Staining 3D collagen gel

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Reflection imaging by scanning confocal is very effective for artificial collagen matrix.  Spaces in the collagen (bubbles or tunnels) or degradation of the collagen can be imaged and quantified.  Works both live and fixed.

We have found that 2nd or 3rd harmonic imaging by multiphoton does not work in artificial collagen matrix.

________________________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: (212) 263-3208  Cell: (914) 309-3270



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Teng Leong Chew
Sent: Thursday, August 30, 2012 3:57 PM
To: [hidden email]
Subject: Staining 3D collagen gel

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

Hi all,

Can anyone recommend a collagen staining protocol that is compatible with fluorescence live cell imaging in 3-D?

We are currently doing cell migration assay and would like to mark the demarcation of the collagen gel (which we make from rat tails) and the culture medium very clearly. In addition, it would be nice to see the tunnels as cells go through the gel as well.

Thanks!!

Regards,
Leong

--
Teng-Leong Chew, PhD

Director, Cell Imaging Facility & Nikon Imaging Center Northwestern University
312-503-2841
Guy Cox-2 Guy Cox-2
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Re: Staining 3D collagen gel

In reply to this post by Teng-Leong Chew
*****
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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****

Why not just look at the second harmonic image from the collagen?  It'll be weaker than from fibrillar collagen but for your purposes I'd think it would work.

                                            Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox  2nd edition 2012  CRC Press
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176     Fax +61 2 9351 7682
             Mobile 0413 281 861
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Teng Leong Chew
Sent: Friday, 31 August 2012 5:57 AM
To: [hidden email]
Subject: Staining 3D collagen gel

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

Hi all,

Can anyone recommend a collagen staining protocol that is
compatible with fluorescence live cell imaging in 3-D?

We are currently doing cell migration assay and would like
to mark the demarcation of the collagen gel (which we make
from rat tails) and the culture medium very clearly. In
addition, it would be nice to see the tunnels as cells
go through the gel as well.

Thanks!!

Regards,
Leong

--
Teng-Leong Chew, PhD

Director, Cell Imaging Facility & Nikon Imaging Center
Northwestern University
312-503-2841
Armstrong, Brian Armstrong, Brian
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Re: Staining 3D collagen gel

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*****

Hello, there was a paper describing SHG imaging of "tracks or tunnels" made by migrating cells in collagen. Since SHG is rather cost efficient we tried this technique, but without much luck.
We also tried reflective imaging on an CLSM and were able to produce images, but they are not very pleasing to the eye.
You can also label collagen with a dye such as Sirius Red or Direct Red 80 for "regular" Confocal (or widefield fluorescent) imaging.
Cheers,

Brian D Armstrong PhD
Assistant Research Professor
Director, Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872

http://www.cityofhope.org/research/support/Light-Microscopy-Digital-Imaging/Pages/default.aspx


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Thursday, August 30, 2012 3:45 PM
To: [hidden email]
Subject: Re: Staining 3D collagen gel

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

Why not just look at the second harmonic image from the collagen?  It'll be weaker than from fibrillar collagen but for your purposes I'd think it would work.

                                            Guy

Optical Imaging Techniques in Cell Biology
by Guy Cox  2nd edition 2012  CRC Press
     http://www.guycox.com/optical.htm
______________________________________________
Guy Cox, MA, DPhil(Oxon), Honorary Associate,
Australian Centre for Microscopy & Microanalysis,
Madsen Building F09, University of Sydney, NSW 2006

Phone +61 2 9351 3176     Fax +61 2 9351 7682
             Mobile 0413 281 861
______________________________________________
      http://www.guycox.net
 


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Teng Leong Chew
Sent: Friday, 31 August 2012 5:57 AM
To: [hidden email]
Subject: Staining 3D collagen gel

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

Hi all,

Can anyone recommend a collagen staining protocol that is
compatible with fluorescence live cell imaging in 3-D?

We are currently doing cell migration assay and would like
to mark the demarcation of the collagen gel (which we make
from rat tails) and the culture medium very clearly. In
addition, it would be nice to see the tunnels as cells
go through the gel as well.

Thanks!!

Regards,
Leong

--
Teng-Leong Chew, PhD

Director, Cell Imaging Facility & Nikon Imaging Center
Northwestern University
312-503-2841


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Steffen Dietzel Steffen Dietzel
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Re: Staining 3D collagen gel

In reply to this post by mcammer
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In contrast to Michaels experience, SHG and THG of collagen gels works
for us. SHG images of such a gel can be found in Figure 2 in

Signal improvement in multiphoton microscopy by reflection with simple
mirrors near the sample
Markus Rehberg ; Fritz Krombach ; Ulrich Pohl ; Steffen Dietzel
J. Biomed. Opt. 15(2), 026017 (April 20, 2010). doi:10.1117/1.3374337

http://biomedicaloptics.spiedigitallibrary.org/article.aspx?articleid=1103350#f2


But you do need quite a bit of output power to achieve a good signal, it
may well be in a range that could be harmfull for cells, we didn't try
that.

cheers

Steffen

On 30.08.2012 23:37, Cammer, Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Reflection imaging by scanning confocal is very effective for artificial collagen matrix.  Spaces in the collagen (bubbles or tunnels) or degradation of the collagen can be imaged and quantified.  Works both live and fixed.
>
> We have found that 2nd or 3rd harmonic imaging by multiphoton does not work in artificial collagen matrix.
>
> ________________________________________________________
> Michael Cammer, Assistant Research Scientist
> Skirball Institute of Biomolecular Medicine
> Lab: (212) 263-3208  Cell: (914) 309-3270
>
>


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

Mail room:
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