Question about surfactants to break surface tension

[Sean Speese-2]

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Hi all,
   Bit of an off topic question, but we do lots of immunos on small bits of
Drosophila tissue that always float on top of the fix due to the surface
tension.  We can use low amounts of triton (.01%) to overcome this, but I
would like to be able to leave detergents out until I am convinced the
fixation is good.  In some cases I would like to do staining of only things
on the cell surface, and thus need to leave detergents completely out of the
staining procedure.  Therefore, does anyone know of a good surfactant that
would break the surface tension but not permeabilize cells?

Thanks,
   Sean Speese

[Boswell, Carl A - (cboswell)]

Hi Sean,
Don't know of an alternative that can do what you want.  I would think that the same properties of a surfactant that make it one would also disturb lipid layers.  

Aside from that, I would caution against the assumption that fixation without detergent allows only exterior staining.  The fixation process will also open holes in membrane and allow Ab to penetrate.  At least test it before committing to that route.  The best result I've had restricting immunostaining to a surface is to stain live material, then fix after washing.
C

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX [phone]



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sean Speese
Sent: Tuesday, January 04, 2011 5:44 PM
To: [hidden email]
Subject: Question about surfactants to break surface tension

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Hi all,
   Bit of an off topic question, but we do lots of immunos on small bits of Drosophila tissue that always float on top of the fix due to the surface tension.  We can use low amounts of triton (.01%) to overcome this, but I would like to be able to leave detergents out until I am convinced the fixation is good.  In some cases I would like to do staining of only things on the cell surface, and thus need to leave detergents completely out of the staining procedure.  Therefore, does anyone know of a good surfactant that would break the surface tension but not permeabilize cells?

Thanks,
   Sean Speese

[Sean Speese-2]

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Hi Carl,
   Point taken regarding the exterior staining, thanks.  My fear is that you
are right about the properties of surfactants.

Sean

On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) <
[hidden email]> wrote:

> Hi Sean,
> Don't know of an alternative that can do what you want.  I would think that
> the same properties of a surfactant that make it one would also disturb
> lipid layers.
>
> Aside from that, I would caution against the assumption that fixation
> without detergent allows only exterior staining.  The fixation process will
> also open holes in membrane and allow Ab to penetrate.  At least test it
> before committing to that route.  The best result I've had restricting
> immunostaining to a surface is to stain live material, then fix after
> washing.
> C
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> Univ. of Arizona
> 520-954-7053
> FAX [phone]
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Sean Speese
> Sent: Tuesday, January 04, 2011 5:44 PM
> To: [hidden email]
> Subject: Question about surfactants to break surface tension
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>   Bit of an off topic question, but we do lots of immunos on small bits of
> Drosophila tissue that always float on top of the fix due to the surface
> tension.  We can use low amounts of triton (.01%) to overcome this, but I
> would like to be able to leave detergents out until I am convinced the
> fixation is good.  In some cases I would like to do staining of only things
> on the cell surface, and thus need to leave detergents completely out of the
> staining procedure.  Therefore, does anyone know of a good surfactant that
> would break the surface tension but not permeabilize cells?
>
> Thanks,
>   Sean Speese
>
>

[Stephen Cody-2]

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G'day Sean,

You indicated the Drosophila tissue bits float on top of the fixative
without triton? In which case it is difficult to see how the fixative will
be able to have much of an effect on anything. I'd be sticking with the
Triton.

Steve
Stephen H. Cody

On 5 January 2011 12:07, Sean Speese <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Carl,
>   Point taken regarding the exterior staining, thanks.  My fear is that you
> are right about the properties of surfactants.
>
> Sean
>
> On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) <
> [hidden email]> wrote:
>
> > Hi Sean,
> > Don't know of an alternative that can do what you want.  I would think
> that
> > the same properties of a surfactant that make it one would also disturb
> > lipid layers.
> >
> > Aside from that, I would caution against the assumption that fixation
> > without detergent allows only exterior staining.  The fixation process
> will
> > also open holes in membrane and allow Ab to penetrate.  At least test it
> > before committing to that route.  The best result I've had restricting
> > immunostaining to a surface is to stain live material, then fix after
> > washing.
> > C
> >
> > Carl A. Boswell, Ph.D.
> > Molecular and Cellular Biology
> > Univ. of Arizona
> > 520-954-7053
> > FAX [phone]
> >
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> > On Behalf Of Sean Speese
> > Sent: Tuesday, January 04, 2011 5:44 PM
> > To: [hidden email]
> > Subject: Question about surfactants to break surface tension
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi all,
> >   Bit of an off topic question, but we do lots of immunos on small bits
> of
> > Drosophila tissue that always float on top of the fix due to the surface
> > tension.  We can use low amounts of triton (.01%) to overcome this, but I
> > would like to be able to leave detergents out until I am convinced the
> > fixation is good.  In some cases I would like to do staining of only
> things
> > on the cell surface, and thus need to leave detergents completely out of
> the
> > staining procedure.  Therefore, does anyone know of a good surfactant
> that
> > would break the surface tension but not permeabilize cells?
> >
> > Thanks,
> >   Sean Speese
> >
>

[Sean Speese-2]

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Yes, floating is a problem cause the fixative is not likely getting in and
penetrating like one would want it to.  However, I want to minimize any
extraction of cytoplasmic components, which is why I was trying to avoid
triton. In particular we are trying to detect if a protein is more abundant
in the cytoplasm after certain treatments, but if having triton present
before fixation is "complete" leads to extraction of certain cytoplasmic
proteins, this could be a problem.

Sean

On Tue, Jan 4, 2011 at 5:23 PM, Stephen Cody <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> G'day Sean,
>
> You indicated the Drosophila tissue bits float on top of the fixative
> without triton? In which case it is difficult to see how the fixative will
> be able to have much of an effect on anything. I'd be sticking with the
> Triton.
>
> Steve
> Stephen H. Cody
>
> On 5 January 2011 12:07, Sean Speese <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Carl,
> >   Point taken regarding the exterior staining, thanks.  My fear is that
> you
> > are right about the properties of surfactants.
> >
> > Sean
> >
> > On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) <
> > [hidden email]> wrote:
> >
> > > Hi Sean,
> > > Don't know of an alternative that can do what you want.  I would think
> > that
> > > the same properties of a surfactant that make it one would also disturb
> > > lipid layers.
> > >
> > > Aside from that, I would caution against the assumption that fixation
> > > without detergent allows only exterior staining.  The fixation process
> > will
> > > also open holes in membrane and allow Ab to penetrate.  At least test
> it
> > > before committing to that route.  The best result I've had restricting
> > > immunostaining to a surface is to stain live material, then fix after
> > > washing.
> > > C
> > >
> > > Carl A. Boswell, Ph.D.
> > > Molecular and Cellular Biology
> > > Univ. of Arizona
> > > 520-954-7053
> > > FAX [phone]
> > >
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List [mailto:
> [hidden email]]
> > > On Behalf Of Sean Speese
> > > Sent: Tuesday, January 04, 2011 5:44 PM
> > > To: [hidden email]
> > > Subject: Question about surfactants to break surface tension
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Hi all,
> > >   Bit of an off topic question, but we do lots of immunos on small bits
> > of
> > > Drosophila tissue that always float on top of the fix due to the
> surface
> > > tension.  We can use low amounts of triton (.01%) to overcome this, but
> I
> > > would like to be able to leave detergents out until I am convinced the
> > > fixation is good.  In some cases I would like to do staining of only
> > things
> > > on the cell surface, and thus need to leave detergents completely out
> of
> > the
> > > staining procedure.  Therefore, does anyone know of a good surfactant
> > that
> > > would break the surface tension but not permeabilize cells?
> > >
> > > Thanks,
> > >   Sean Speese
> > >
> >
>

[Ray Gilbert]

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Is the floating caused by the low density of your sample or air trapped in the tissue? If it is the latter you could try putting it in a small jar with an airtight lid then extracting the air using a syringe and needle. I used to do this with lung tissue many moons ago when I was doing EM. The tissue sank very well, something it didn't do otherwise. I can give more details off thread if you need them.

Thanks

Ray Gilbert
Laboratory Manager
Centre for Brain Research |5th Floor |  FMHS
University of Auckland
Private Bag 92019 |Auckland Mailing Centre |Auckland 1142 |New Zealand

ph [phone] |fax + 64 9 373 8774 |mob [phone] |speed dial 60198 (UOA only)
[hidden email]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sean Speese
Sent: Wednesday, 5 January 2011 2:55 p.m.
To: [hidden email]
Subject: Re: Question about surfactants to break surface tension

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Yes, floating is a problem cause the fixative is not likely getting in and
penetrating like one would want it to.  However, I want to minimize any
extraction of cytoplasmic components, which is why I was trying to avoid
triton. In particular we are trying to detect if a protein is more abundant
in the cytoplasm after certain treatments, but if having triton present
before fixation is "complete" leads to extraction of certain cytoplasmic
proteins, this could be a problem.

Sean

On Tue, Jan 4, 2011 at 5:23 PM, Stephen Cody <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> G'day Sean,
>
> You indicated the Drosophila tissue bits float on top of the fixative
> without triton? In which case it is difficult to see how the fixative will
> be able to have much of an effect on anything. I'd be sticking with the
> Triton.
>
> Steve
> Stephen H. Cody
>
> On 5 January 2011 12:07, Sean Speese <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Carl,
> >   Point taken regarding the exterior staining, thanks.  My fear is that
> you
> > are right about the properties of surfactants.
> >
> > Sean
> >
> > On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) <
> > [hidden email]> wrote:
> >
> > > Hi Sean,
> > > Don't know of an alternative that can do what you want.  I would think
> > that
> > > the same properties of a surfactant that make it one would also disturb
> > > lipid layers.
> > >
> > > Aside from that, I would caution against the assumption that fixation
> > > without detergent allows only exterior staining.  The fixation process
> > will
> > > also open holes in membrane and allow Ab to penetrate.  At least test
> it
> > > before committing to that route.  The best result I've had restricting
> > > immunostaining to a surface is to stain live material, then fix after
> > > washing.
> > > C
> > >
> > > Carl A. Boswell, Ph.D.
> > > Molecular and Cellular Biology
> > > Univ. of Arizona
> > > 520-954-7053
> > > FAX [phone]
> > >
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List [mailto:
> [hidden email]]
> > > On Behalf Of Sean Speese
> > > Sent: Tuesday, January 04, 2011 5:44 PM
> > > To: [hidden email]
> > > Subject: Question about surfactants to break surface tension
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Hi all,
> > >   Bit of an off topic question, but we do lots of immunos on small bits
> > of
> > > Drosophila tissue that always float on top of the fix due to the
> surface
> > > tension.  We can use low amounts of triton (.01%) to overcome this, but
> I
> > > would like to be able to leave detergents out until I am convinced the
> > > fixation is good.  In some cases I would like to do staining of only
> > things
> > > on the cell surface, and thus need to leave detergents completely out
> of
> > the
> > > staining procedure.  Therefore, does anyone know of a good surfactant
> > that
> > > would break the surface tension but not permeabilize cells?
> > >
> > > Thanks,
> > >   Sean Speese
> > >
> >
>

[Tobias Baskin]

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Hi Sean,
        One thing you can contemplate is doing it mechanically. We
have good results processing small and floaty roots of the plant
Arabidopsis thaliana by sandwiching them between two Formvar films.
The films are over a wire loop which definitely sinks. Formvar is
strong but allows all sorts of stuff to cross. But not antibodies.
You have to take the tissue out from the films for that.

        If this sounds useful, I can give more details.

        Good luck,
                Tobias

>
>
>Hi all,
>    Bit of an off topic question, but we do lots of immunos on small bits of
>Drosophila tissue that always float on top of the fix due to the surface
>tension.  We can use low amounts of triton (.01%) to overcome this, but I
>would like to be able to leave detergents out until I am convinced the
>fixation is good.  In some cases I would like to do staining of only things
>on the cell surface, and thus need to leave detergents completely out of the
>staining procedure.  Therefore, does anyone know of a good surfactant that
>would break the surface tension but not permeabilize cells?
>
>Thanks,
>    Sean Speese

--
       _      ____          __   ____  
      /  \   /          / \    /   \ \        Tobias I. Baskin
     /   /  /          /   \   \      \         Biology Department
    /_ /   __      /__ \   \       \__    611 N. Pleasant St.
   /      /          /       \   \       \        University of Massachusetts
  /      /          /         \   \       \    Amherst, MA, 01003
/      / ___   /           \   \__/  \ ____
www.bio.umass.edu/biology/baskin
Voice: 413 - 545 - 1533 Fax: 413 - 545 - 3243

[farmer]

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Wanna ask permission of the author?



Dear Speakeasy,
Ah, in my experience nothing breaks the surface tension like a few shots of
Becherovka!  Not sure I can remember doing it with Drosophila but I do
vividly recall the summer of 1905 when Albert E.,  his wife Mileva and I got
totally hammered watching the The Scarlet Pimpernel which had recently
opened at the New Theatre in London.  Albert had just finished working out
how special relativity relates to Brownian motion when I said "Lassen Sie uns
getrunken gehen und erhalten!"  Always one to rise to the occasion  Mileva
challenged me to a game of quarters, which I handily won.  Poor Albert spent
most of the evening in the men's room praying to the porcelain goddess while
Mileva and I danced the night away.  Like you we ultimately ended up
staining some tissue, but I'd better not go there, if you know what I mean.





http://www.einstein-website.de/z_information/faq-e.html
Thus in summer 1905 he wrote in a postcard to Conrad Habicht: "Totally drunk unfortunately both of
us under the table. ...". This is meant to be Einstein and his first wife Mileva.



On 4 Jan 2011 at 18:44, Sean Speese wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy *****
>
> Hi all,
>    Bit of an off topic question, but we do lots of immunos on small
>    bits of
> Drosophila tissue that always float on top of the fix due to the
> surface tension.  We can use low amounts of triton (.01%) to overcome
> this, but I would like to be able to leave detergents out until I am
> convinced the fixation is good.  In some cases I would like to do
> staining of only things on the cell surface, and thus need to leave
> detergents completely out of the staining procedure.  Therefore, does
> anyone know of a good surfactant that would break the surface tension
> but not permeabilize cells?
>
> Thanks,
>    Sean Speese
>
> --
> This message has been scanned for viruses and
> dangerous content by MailScanner, and is
> believed to be clean.
>

Mark Farmer
Cellular Biology
University of Georgia
Athens, GA 30602 USA
Tel: [phone]
FAX [phone]

--
This message has been scanned for viruses and
dangerous content by MailScanner, and is
believed to be clean.

[Cameron Nowell]

Hi Sean,

In a past life when i worked with Drosophila a lot we used to do it all in 1.5ml tubes and just put them on a nutating platform (like this http://www.globescientific.com/nutating-mixer-ii-large-platform-3d-tube-rotator-522-p-2306.html) . It kept everything mixing fine and was very gentle on the tissues being fixed (even kept lymph glands whole without a problem). It laso works well for staining as you get nice constant coverage with your antibody.

Cheers

Cam



Cameron J. Nowell
Microscopy Manager
Centre for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008
Royal Melbourne Hospital
Victoria, 3050
AUSTRALIA
Office: [phone]
Mobile: [phone]
Fax: [phone]
Facility Website



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sean Speese
Sent: Wednesday, 5 January 2011 11:44 AM
To: [hidden email]
Subject: Question about surfactants to break surface tension

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Hi all,
   Bit of an off topic question, but we do lots of immunos on small bits of
Drosophila tissue that always float on top of the fix due to the surface
tension.  We can use low amounts of triton (.01%) to overcome this, but I
would like to be able to leave detergents out until I am convinced the
fixation is good.  In some cases I would like to do staining of only things
on the cell surface, and thus need to leave detergents completely out of the
staining procedure.  Therefore, does anyone know of a good surfactant that
would break the surface tension but not permeabilize cells?

Thanks,
   Sean Speese

[Sally Stowe]

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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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Hi Sean,
might be worth trying a little bit of DMSO - dipping an applicator
stick soaked in DMSO into  the fix may be enough.
cheers
Sally

Sally Stowe
ANU



On 5 January 2011 12:55, Sean Speese <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Yes, floating is a problem cause the fixative is not likely getting in and
> penetrating like one would want it to.  However, I want to minimize any
> extraction of cytoplasmic components, which is why I was trying to avoid
> triton. In particular we are trying to detect if a protein is more abundant
> in the cytoplasm after certain treatments, but if having triton present
> before fixation is "complete" leads to extraction of certain cytoplasmic
> proteins, this could be a problem.
>
> Sean
>
> On Tue, Jan 4, 2011 at 5:23 PM, Stephen Cody <[hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> G'day Sean,
>>
>> You indicated the Drosophila tissue bits float on top of the fixative
>> without triton? In which case it is difficult to see how the fixative will
>> be able to have much of an effect on anything. I'd be sticking with the
>> Triton.
>>
>> Steve
>> Stephen H. Cody
>>
>> On 5 January 2011 12:07, Sean Speese <[hidden email]> wrote:
>>
>> > *****
>> > To join, leave or search the confocal microscopy listserv, go to:
>> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > *****
>> >
>> > Hi Carl,
>> >   Point taken regarding the exterior staining, thanks.  My fear is that
>> you
>> > are right about the properties of surfactants.
>> >
>> > Sean
>> >
>> > On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) <
>> > [hidden email]> wrote:
>> >
>> > > Hi Sean,
>> > > Don't know of an alternative that can do what you want.  I would think
>> > that
>> > > the same properties of a surfactant that make it one would also disturb
>> > > lipid layers.
>> > >
>> > > Aside from that, I would caution against the assumption that fixation
>> > > without detergent allows only exterior staining.  The fixation process
>> > will
>> > > also open holes in membrane and allow Ab to penetrate.  At least test
>> it
>> > > before committing to that route.  The best result I've had restricting
>> > > immunostaining to a surface is to stain live material, then fix after
>> > > washing.
>> > > C
>> > >
>> > > Carl A. Boswell, Ph.D.
>> > > Molecular and Cellular Biology
>> > > Univ. of Arizona
>> > > 520-954-7053
>> > > FAX [phone]
>> > >
>> > >
>> > >
>> > > -----Original Message-----
>> > > From: Confocal Microscopy List [mailto:
>> [hidden email]]
>> > > On Behalf Of Sean Speese
>> > > Sent: Tuesday, January 04, 2011 5:44 PM
>> > > To: [hidden email]
>> > > Subject: Question about surfactants to break surface tension
>> > >
>> > > *****
>> > > To join, leave or search the confocal microscopy listserv, go to:
>> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> > > *****
>> > >
>> > > Hi all,
>> > >   Bit of an off topic question, but we do lots of immunos on small bits
>> > of
>> > > Drosophila tissue that always float on top of the fix due to the
>> surface
>> > > tension.  We can use low amounts of triton (.01%) to overcome this, but
>> I
>> > > would like to be able to leave detergents out until I am convinced the
>> > > fixation is good.  In some cases I would like to do staining of only
>> > things
>> > > on the cell surface, and thus need to leave detergents completely out
>> of
>> > the
>> > > staining procedure.  Therefore, does anyone know of a good surfactant
>> > that
>> > > would break the surface tension but not permeabilize cells?
>> > >
>> > > Thanks,
>> > >   Sean Speese
>> > >
>> >
>>
>

[Paul Rigby-2]

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*****

Sean,
Have you thought about trying to fix your Drosophila with a vapour? If you could do this you might minimise the risks of losing some of your intracellular components. I have never seen vapour fixation described for immunostaining but the electron microscopy people used to do this (at least many years ago when I was learning the trade) using glutaraldehyde or osmium vapour. I would not even try either of these fixatives, but it might work with a formaldehyde vapour. A little gentle heating of a formaldehyde solution in a closed container (make sure you do this in a fume hood) might help vaporise the fixative enough to allow the gas to fix your drosophila if they are suspended on a mesh above the liquid.

Also, I concur with Carl - almost all fixatives I have used will punch holes through cell membranes (at least big enough for antibodies to penetrate). I suspect that Triton and saponin simply punch bigger holes.

Hope this helps.
Cheers
Paul Rigby


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 Sean Speese
Sent: Wednesday, 5 January 2011 9:55 AM
To: [hidden email]
Subject: Re: Question about surfactants to break surface tension

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Yes, floating is a problem cause the fixative is not likely getting in and
penetrating like one would want it to.  However, I want to minimize any
extraction of cytoplasmic components, which is why I was trying to avoid
triton. In particular we are trying to detect if a protein is more abundant
in the cytoplasm after certain treatments, but if having triton present
before fixation is "complete" leads to extraction of certain cytoplasmic
proteins, this could be a problem.

Sean

On Tue, Jan 4, 2011 at 5:23 PM, Stephen Cody <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> G'day Sean,
>
> You indicated the Drosophila tissue bits float on top of the fixative
> without triton? In which case it is difficult to see how the fixative will
> be able to have much of an effect on anything. I'd be sticking with the
> Triton.
>
> Steve
> Stephen H. Cody
>
> On 5 January 2011 12:07, Sean Speese <[hidden email]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi Carl,
> >   Point taken regarding the exterior staining, thanks.  My fear is that
> you
> > are right about the properties of surfactants.
> >
> > Sean
> >
> > On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) <
> > [hidden email]> wrote:
> >
> > > Hi Sean,
> > > Don't know of an alternative that can do what you want.  I would think
> > that
> > > the same properties of a surfactant that make it one would also disturb
> > > lipid layers.
> > >
> > > Aside from that, I would caution against the assumption that fixation
> > > without detergent allows only exterior staining.  The fixation process
> > will
> > > also open holes in membrane and allow Ab to penetrate.  At least test
> it
> > > before committing to that route.  The best result I've had restricting
> > > immunostaining to a surface is to stain live material, then fix after
> > > washing.
> > > C
> > >
> > > Carl A. Boswell, Ph.D.
> > > Molecular and Cellular Biology
> > > Univ. of Arizona
> > > 520-954-7053
> > > FAX [phone]
> > >
> > >
> > >
> > > -----Original Message-----
> > > From: Confocal Microscopy List [mailto:
> [hidden email]]
> > > On Behalf Of Sean Speese
> > > Sent: Tuesday, January 04, 2011 5:44 PM
> > > To: [hidden email]
> > > Subject: Question about surfactants to break surface tension
> > >
> > > *****
> > > To join, leave or search the confocal microscopy listserv, go to:
> > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > > *****
> > >
> > > Hi all,
> > >   Bit of an off topic question, but we do lots of immunos on small bits
> > of
> > > Drosophila tissue that always float on top of the fix due to the
> surface
> > > tension.  We can use low amounts of triton (.01%) to overcome this, but
> I
> > > would like to be able to leave detergents out until I am convinced the
> > > fixation is good.  In some cases I would like to do staining of only
> > things
> > > on the cell surface, and thus need to leave detergents completely out
> of
> > the
> > > staining procedure.  Therefore, does anyone know of a good surfactant
> > that
> > > would break the surface tension but not permeabilize cells?
> > >
> > > Thanks,
> > >   Sean Speese
> > >
> >
>

[Oshel, Philip Eugene]

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

i've used cotton plugs for this. Shove the cotton into the tube, down
into the fixative - the cotton wicks up fix and physically holds the
sample submerged in the fixative.
But I also suspect air in the trachea is what's keeping the tissue up
(if there is no exoskeleton on the samples, then I'd suspect waxy
lipids on the cuticle), so pulling a gentle vacuum should work. You
might have to use some vacuum/vent/vacuum/vent cycles.

Phil

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Hi all,
>    Bit of an off topic question, but we do lots of immunos on small bits of
>Drosophila tissue that always float on top of the fix due to the surface
>tension.  We can use low amounts of triton (.01%) to overcome this, but I
>would like to be able to leave detergents out until I am convinced the
>fixation is good.  In some cases I would like to do staining of only things
>on the cell surface, and thus need to leave detergents completely out of the
>staining procedure.  Therefore, does anyone know of a good surfactant that
>would break the surface tension but not permeabilize cells?
>
>Thanks,
>    Sean Speese

--
Philip Oshel
Microscopy Facility Supervisor
Biology Department
024C Brooks Hall
Central Michigan University
Mt. Pleasant, MI 48859
[phone]

[Sean Speese-2]

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

Hi Carl,
  I just wanted to follow up with a few more questions about staining
epitopes just at the surface.  My assumption was that the little bit of
permeabilization that one can get from para may occur if you use 37%
formaldehyde as your starting solution, as it has some low level of
methanol.  However, if I understand you correctly, even high grade EM para
will lead to some permeabilization? Is this due cells being osmotically
shocked and lysed if the osmolarity is off, or some other mechanism?

 I would like to stain Drosophila brains to see if certain proteins are
secreted into the ECM by glia and neurons after injury.  I could stain live
material, but dissecting open the live brain is essentially going to elicit
some type of injury response, thus making it hard to have a clean control.
Maybe I could keep the live brains that I am staining at 4 degrees to
minimize any reaction from the dissection itself.

Thanks,
 Sean

On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) <
[hidden email]> wrote:

> Hi Sean,
> Don't know of an alternative that can do what you want.  I would think that
> the same properties of a surfactant that make it one would also disturb
> lipid layers.
>
> Aside from that, I would caution against the assumption that fixation
> without detergent allows only exterior staining.  The fixation process will
> also open holes in membrane and allow Ab to penetrate.  At least test it
> before committing to that route.  The best result I've had restricting
> immunostaining to a surface is to stain live material, then fix after
> washing.
> C
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> Univ. of Arizona
> 520-954-7053
> FAX [phone]
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Sean Speese
> Sent: Tuesday, January 04, 2011 5:44 PM
> To: [hidden email]
> Subject: Question about surfactants to break surface tension
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>   Bit of an off topic question, but we do lots of immunos on small bits of
> Drosophila tissue that always float on top of the fix due to the surface
> tension.  We can use low amounts of triton (.01%) to overcome this, but I
> would like to be able to leave detergents out until I am convinced the
> fixation is good.  In some cases I would like to do staining of only things
> on the cell surface, and thus need to leave detergents completely out of the
> staining procedure.  Therefore, does anyone know of a good surfactant that
> would break the surface tension but not permeabilize cells?
>
> Thanks,
>   Sean Speese
>
>

[Mario]

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

Sean,

Just to add to Paul's and Carl's comments, vapor
based fixation is worthy of consideration. You
might consider a compartment that allows you to
gently heat some paraformaldehyde solid in the
presence of your sample. The formaldehyde vapor
then fixes your specimen. As mentioned by Paul,
osmium tetroxide fixation is a possibility but
acts on the membrane lipids, which is probably
bad leaving them permeable.

Detergents being amphipathic almost by definition
must perturb the cell bilayer; however, they are
not all the same. Triton was demonstrated to
permeabilize bilayers somewhat selectively for
sodium over potassium. I may have that backwards,
but in either case this indicates that detergent
induced defects can be quite small, ~ the size of
a hydrated K+ ion. Most cells if permeabilized in
this way will become osmotically unstable and
break open with rather larger holes. Triton is
definitely not a good way to retain internal
proteins/targets if used before fixation. Tween
20 is a gentler alternative. The problem, of
course, is that to get immersion you might end up
having to use a concentration high enough that
causes a similar cell membrane disruption. In any
case, it might be worth trying different
detergents with some regard for their CMCs, chain
lengths, aromatic components, and any net charge
they might carry. Again, not all detergents are
the same.

As an alternative, it might be worth trying PEG
(3 to 8 KDa) as a surfactant. I have never
measured the difference between PBS ± PEG but a
few percent 8 KDa PEG might provide sufficient
slipperiness to allow submersion without busting
open your cells. They might even shrink if you
don't compensate for the difference in
osmolality. You'd have to consider whether PEG
would compromise your labeling protocol in some
other way.

Let us know what works,
Mario

>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Sean,
>Have you thought about trying to fix your
>Drosophila with a vapour? If you could do this
>you might minimise the risks of losing some of
>your intracellular components. I have never seen
>vapour fixation described for immunostaining but
>the electron microscopy people used to do this
>(at least many years ago when I was learning the
>trade) using glutaraldehyde or osmium vapour. I
>would not even try either of these fixatives,
>but it might work with a formaldehyde vapour. A
>little gentle heating of a formaldehyde solution
>in a closed container (make sure you do this in
>a fume hood) might help vaporise the fixative
>enough to allow the gas to fix your drosophila
>if they are suspended on a mesh above the liquid.
>
>Also, I concur with Carl - almost all fixatives
>I have used will punch holes through cell
>membranes (at least big enough for antibodies to
>penetrate). I suspect that Triton and saponin
>simply punch bigger holes.
>
>Hope this helps.
>Cheers
>Paul Rigby
>
>
>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 Sean Speese
>Sent: Wednesday, 5 January 2011 9:55 AM
>To: [hidden email]
>Subject: Re: Question about surfactants to break surface tension
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
>http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>*****
>
>Yes, floating is a problem cause the fixative is not likely getting in and
>penetrating like one would want it to.  However, I want to minimize any
>extraction of cytoplasmic components, which is why I was trying to avoid
>triton. In particular we are trying to detect if a protein is more abundant
>in the cytoplasm after certain treatments, but if having triton present
>before fixation is "complete" leads to extraction of certain cytoplasmic
>proteins, this could be a problem.
>
>Sean
>
>On Tue, Jan 4, 2011 at 5:23 PM, Stephen Cody <[hidden email]> wrote:
>
>>  *****
>>  To join, leave or search the confocal microscopy listserv, go to:
>>  http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  *****
>>
>>  G'day Sean,
>>
>>  You indicated the Drosophila tissue bits float on top of the fixative
>>  without triton? In which case it is difficult to see how the fixative will
>>  be able to have much of an effect on anything. I'd be sticking with the
>>  Triton.
>>
>>  Steve
>>  Stephen H. Cody
>>
>>  On 5 January 2011 12:07, Sean Speese <[hidden email]> wrote:
>>
>>  > *****
>>  > To join, leave or search the confocal microscopy listserv, go to:
>>  > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  > *****
>>  >
>>  > Hi Carl,
>>  >   Point taken regarding the exterior staining, thanks.  My fear is that
>>  you
>>  > are right about the properties of surfactants.
>>  >
>>  > Sean
>>  >
>>  > On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) <
>>  > [hidden email]> wrote:
>>  >
>>  > > Hi Sean,
>>  > > Don't know of an alternative that can do what you want.  I would think
>>  > that
>>  > > the same properties of a surfactant that make it one would also disturb
>>  > > lipid layers.
>>  > >
>>  > > Aside from that, I would caution against the assumption that fixation
>>  > > without detergent allows only exterior staining.  The fixation process
>>  > will
>>  > > also open holes in membrane and allow Ab to penetrate.  At least test
>>  it
>>  > > before committing to that route.  The best result I've had restricting
>>  > > immunostaining to a surface is to stain live material, then fix after
>>  > > washing.
>>  > > C
>>  > >
>>  > > Carl A. Boswell, Ph.D.
>>  > > Molecular and Cellular Biology
>>  > > Univ. of Arizona
>>  > > 520-954-7053
>>  > > FAX [phone]
>>  > >
>>  > >
>>  > >
>>  > > -----Original Message-----
>>  > > From: Confocal Microscopy List [mailto:
>>  [hidden email]]
>>  > > On Behalf Of Sean Speese
>>  > > Sent: Tuesday, January 04, 2011 5:44 PM
>>  > > To: [hidden email]
>>  > > Subject: Question about surfactants to break surface tension
>>  > >
>>  > > *****
>>  > > To join, leave or search the confocal microscopy listserv, go to:
>>  > > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>  > > *****
>>  > >
>>  > > Hi all,
>>  > >   Bit of an off topic question, but we do lots of immunos on small bits
>>  > of
>>  > > Drosophila tissue that always float on top of the fix due to the
>>  surface
>>  > > tension.  We can use low amounts of triton (.01%) to overcome this, but
>>  I
>>  > > would like to be able to leave detergents out until I am convinced the
>>  > > fixation is good.  In some cases I would like to do staining of only
>>  > things
>>  > > on the cell surface, and thus need to leave detergents completely out
>>  of
>>  > the
>>  > > staining procedure.  Therefore, does anyone know of a good surfactant
>>  > that
>>  > > would break the surface tension but not permeabilize cells?
>>  > >
>>  > > Thanks,
>>  > >   Sean Speese
>>  > >
>>  >
>>

--
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[hidden email]
[hidden email]

[Boswell, Carl A - (cboswell)]

Hi Sean,
Sorry for the delay.  

All the staining results I"ve referred to used 3% formaldehyde (before or after Ab treatment) which was made from powder no more than an hour before use.   I can't say what the mechanism of leaking is, but osmolarity and pH were specifically controlled for.  My guess is that the cross-linking, hence denaturation, of membrane proteins disrupts  the normal interaction between lipids and those proteins, and provides an access for larger molecules across the barrier.

I may not be clear on your procedure.  If you dissect the brains, you would expect an injury response, hence the problem with obtaining a good negative control.  When you refer to "live brains", is that tissue still in the animal?

Overnight incubation of sample with Ab at 4C is a standard procedure in many cases and can give you good results.  The problem I see here is if you get good penetration of the Ab into the tissue, you will need to do substantial washing before fixing and adding secondary Ab.  That may preclude having live material in the end.

Please feel free to correspond off list.
Cheers,
C


Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
Univ. of Arizona
520-954-7053
FAX [phone]


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Sean Speese
Sent: Wednesday, January 05, 2011 10:50 AM
To: [hidden email]
Subject: Re: Question about surfactants to break surface tension

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

Hi Carl,
  I just wanted to follow up with a few more questions about staining epitopes just at the surface.  My assumption was that the little bit of permeabilization that one can get from para may occur if you use 37% formaldehyde as your starting solution, as it has some low level of methanol.  However, if I understand you correctly, even high grade EM para will lead to some permeabilization? Is this due cells being osmotically shocked and lysed if the osmolarity is off, or some other mechanism?

 I would like to stain Drosophila brains to see if certain proteins are secreted into the ECM by glia and neurons after injury.  I could stain live material, but dissecting open the live brain is essentially going to elicit some type of injury response, thus making it hard to have a clean control.
Maybe I could keep the live brains that I am staining at 4 degrees to minimize any reaction from the dissection itself.

Thanks,
 Sean

On Tue, Jan 4, 2011 at 4:59 PM, Boswell, Carl A - (cboswell) < [hidden email]> wrote:

> Hi Sean,
> Don't know of an alternative that can do what you want.  I would think
> that the same properties of a surfactant that make it one would also
> disturb lipid layers.
>
> Aside from that, I would caution against the assumption that fixation
> without detergent allows only exterior staining.  The fixation process
> will also open holes in membrane and allow Ab to penetrate.  At least
> test it before committing to that route.  The best result I've had
> restricting immunostaining to a surface is to stain live material,
> then fix after washing.
> C
>
> Carl A. Boswell, Ph.D.
> Molecular and Cellular Biology
> Univ. of Arizona
> 520-954-7053
> FAX [phone]
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[hidden email]]
> On Behalf Of Sean Speese
> Sent: Tuesday, January 04, 2011 5:44 PM
> To: [hidden email]
> Subject: Question about surfactants to break surface tension
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi all,
>   Bit of an off topic question, but we do lots of immunos on small
> bits of Drosophila tissue that always float on top of the fix due to
> the surface tension.  We can use low amounts of triton (.01%) to
> overcome this, but I would like to be able to leave detergents out
> until I am convinced the fixation is good.  In some cases I would like
> to do staining of only things on the cell surface, and thus need to
> leave detergents completely out of the staining procedure.  Therefore,
> does anyone know of a good surfactant that would break the surface tension but not permeabilize cells?
>
> Thanks,
>   Sean Speese
>
>