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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:

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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 <[log in to unmask]> wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
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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) <
> > [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
> > >
> >
>