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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


>*****
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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:[log in to unmask]] On 
>Behalf Of Sean Speese
>Sent: Wednesday, 5 January 2011 9:55 AM
>To: [log in to unmask]
>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 <[log in to unmask]> 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 <[log in to unmask]> 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) <
>>  > [log in to unmask]> 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 520-621-3709
>>  > >
>>  > >
>>  > >
>>  > > -----Original Message-----
>>  > > From: Confocal Microscopy List [mailto:
>>  [log in to unmask]]
>>  > > On Behalf Of Sean Speese
>>  > > Sent: Tuesday, January 04, 2011 5:44 PM
>>  > > To: [log in to unmask]
>>  > > 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.

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