12/7/97
I am interested in visualising cellulose microfibrils and
microtubules/microfilaments in wood-forming cells of trees in the confocal
microscope. I use FITC-labelled antibodies for cytoskeleton and would like
to stain the cellulose with a fluorescent dye. If I had access to a UV
laser I would have no hesitation in using calcofluor/tinopal, but I don't.
So, can anybody suggest a visible light-excited fluorochrome that will
localise the cellulose and will permit imaging of both cellulose and
cytoskeleton? (If it helps we have a Zeiss 510 with 488, 568 and 633 nm
lines.)
I thank you in advance,
Yours sincerely,
Nigel Chaffey
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Dr Nigel Chaffey,
Dept Forest Genetics & Plant Physiology,
Swedish University of Agricultural Sciences,
S-901 83 Umeå,
Sweden
Phone: +46-90-786-6305
Fax: +46-90-786-5901
eMail: nigel.chaffey@genfys.slu.se
with Congo Red. In your case this would probably mean you will need an
alternate secondary antibody fluorophore.
hberg@CC.MEMPHIS.EDU
Since you already are immunolabeling the cytoskeleton why not use a
lectin with a tag other than FITC (i.e. TRITC, Texas Red)? I've had
great luck with lectins.
I'm usually looking for chitin, so I don't have a favoirte
cellulose lectin, maybe some else does?
Richard E. Edelmann, Ph.D.
Electron Microscopy Facility Supervisor
352 Pearson Hall
Miami University, Oxford, OH 45056
Ph: 513.529.5712 Fax: 513.529.4243
E-mail: edelmare@muohio.edu
cellulose by binding to it, like congo red? Or even something as venerable
as tol blue or safranin or any other of those old stains? No idea how well
these would fluoresce, or whether there would be too much bleed-through,
but should be relatively quick to try out.
good luck, and I would be interested to hear the results,
cheers,
Rosemary White
Department of Biological Sciences
Monash University, Melbourne, Victoria 3168, Australia
phone 61-3-9905 5670
fax 61-3-9905 5613 email r.g.white@sci.monash.edu.au
ar-ion laser. Cfr Stickens & Verbelen. J.Microscopy some where in 1996
or 1995.
Patrick Van Oostveldt
lab. Biochemistry & Molecular Cytology
Coupure Links 653
B 9000 GENT
tel: 32 9 2645969
fax 32 9 264 6231
I have several suggestions. Try safranine (0.1% aq). This will stain
cellulose green and lignin red. It will also stain the membrane
systems in your cells. Unfortunately this will probably mask any
signal from your FITC label but you could try acquiring the FITC image
first then staining with safranine and try to find the same cell. Make
sure you mount in a non-solvent based medium such as water to avoid
leaching of stain.
My second suggestion is a bit more drastic. After acquiring your FITC
image, treat you section with 1M NaOH, wash, dry, treat with iodine
solution and preciptitate the iodine with conc. nitric acid, wash in
ethanol, mount in ethylene glycol and observe using reflection mode on
your confocal. If you are really lucky you will get an image showing
the cellulose microfibrils (actually bundles of microfibrils). This
probably won't work if your material is unlignified which I assume it
is.
The ideal way of doing this is to have a cellulose specific label such
as cellulase-Cy5 which will give you a label distinct from your FITC
but you would need to make this yourself !
If you get any of these to work let me know as we want to do the same
thing.
Best regards
Lloyd Donaldson
New Zealand Forest Research Institute Ltd
Private Bag 3020 Rotorua
New Zealand
email donaldsl@fri.cri.nz
I don't know about wood but in human tissue we use a .01% Evans Blue as a
total protien counterstain in conjunction with FITC tagged primary. The
evans blue excites and emmits like rodamine or texas red.
Bob
underwoo@u.washington.edu