All good suggestions. I used to mount plant tissues in a home-made well
(hole drilled through glass slide, coverslip glued over hole with valap,
tissue placed onto coverslip in well). Then I'd drop one third of a small
circular coverslip onto each end of the tissue, and glue in place with more
valap - taking care not to touch the tissue with the warm valap. Then the
tissue was tissue anchored down and I could change solutions if needed, and
also microinject from above - on an inverted scope.
As John says, cyanoacrylate glue will work as a fixative and kill the
tissue.
You could also stabilise the actin cytoskeleton with a protein crosslinker,
like MBS, but as John says, if you're trying to image dynamics of a
relatively unperturbed system, modifying the cytoskeleton will probably
affect recovery into the bleached area. Nuclei do move around, especially
if the cells have been perturbed in any way.
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
E [log in to unmask]
On 15/05/10 1:56 AM, "John Runions" <[log in to unmask]> wrote:
> Also, I meant to say -
>
> There are much better ways to immobilize whole tissues and leaf pieces
> than with glue. Try mounting the leaf piece in low-melting temperature
> agarose and then sealing the slide with VALAP (which is vaseline / lanolin
> / paraffin wax mixed 1:1:1 and heated to homogenize. You use a hot metal
> spatula to 'draw' the warm liquid VALAP around the edges of the coverslip
> and then it sets and seals the coverslip without being toxic to living
> cells). Tissue will stay happy and immobilized for hours in this setup.
>
>
> !!!! Don't use Krazy Glue or any solvent based glue on leaves. Even at
> some distance from the cells, this will kill them!!!
>
> I would suggest removing the actin cytoskeleton using, e.g. latrunculinB,
> if nuclear movement is an issue. This, of course depends on what your
> experiment is. If you are trying to observe FRAP of a protein or
> mechanism that transports on microfilaments, then depolymerising the actin
> won't work.
>
> John.
>
>
>
> Dear All,
>
> Thanks you very much for all of your input (comments, suggestions,
> protocols
> etc ) in regard to the photobleaching in our Zeiss510. The good news is
> that
> we got some progress on this but bad news is that a new question needs to
> be
> addressed: the movement of the bleaching subject/organelle.
>
> We are using transiently GFP-expressed tobacco leaves and want to bleach
> whole or partial nucleus. However the nucleus seems to move at lease about
> 1-2um in a 5min period. we tried to use larger pinhole and larger ROI but
> the plane of focus is still an issue (so only saw partial bleach). Zeiss
> specialist suggest to stick the leaves on the slide with krazy glue and
> surrounded by something to avoid the leave floating/vibrating (we are
> using
> inverted microscope). We tried but the movement was still noticeable. Have
> some of you meet a similar problem and how did it solved?
>
> Meantime I am want to install a macro that could track the cell movement
> (developed by Ellenberg). Any experience on it?
>
> Thank you in advance.
> --
> View this message in context:
> http://confocal-microscopy-list.588098.n2.nabble.com/Photobleach-FRAP-problem-
> in-Zeiss-510Meta-tp5038048p5051254.html
> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email: [log in to unmask]
> phone: +44 (0) 1865 483 964
>
> Runions¹ lab web site
> (http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)
>
> Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)
>
> Oxford Brookes Master's in Bioimaging with Molecular Technology
> (http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)
>
>
>
> *********************************
> C. John Runions, Ph.D.
> School of Life Sciences
> Oxford Brookes University
> Oxford, UK
> OX3 0BP
>
> email: [log in to unmask]
> phone: +44 (0) 1865 483 964
>
> Runions¹ lab web site
> (http://www.brookes.ac.uk/lifesci/runions/HTMLpages/index.html!)
>
> Visit The Illuminated Plant Cell (http://www.illuminatedcell.com/ER.html)
>
> Oxford Brookes Master's in Bioimaging with Molecular Technology
> (http://www.brookes.ac.uk/studying/courses/postgraduate/2007/bmt)
|
