Anna
Arabidopsis influorescence stems or short shoots can be embedded in LR White without problem provided you cut the stems in half so they are not completely surrounded in cuticle. I haven't tried leaves - might need vacuum to assist with infiltration. Have you tried confocal on your wax embedded material ?
Regards
Dr Lloyd Donaldson
Senior Scientist, Project Leader - Microscopy/Wood Identification
Scion - Next Generation Biomaterials
Private Bag 3020, Rotorua
New Zealand 3010
Ph: 64 7 343 5581
-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of George McNamara
Sent: Tuesday, 25 January 2011 12:36 a.m.
To: [log in to unmask]
Subject: Re: Embedding Arabidopsis Thaliana for confocal
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Hi Anna,
Only way to know is to do the experiment.
I have a suggestion for transitioning from IHC to fluorescence confocal
microscopy: assuming you are using HRP with DAB + H2O2, you can
substitute DAB with a fluorescent tyramide (available from
Invitrogen/Molecular Probes,
http://probes.invitrogen.com/media/pis/mp20911.pdf ) or PerkinElmer Life
Sciences. You can ignore the other components in the kit (store those
as directed in the kit). Pick a fluorophore in a wavelength range that
has low autofluorescence for your specimen (one of your current fixed
specimens - ex. a negative control). I strongly encourage the use of
serial sections, with alternating sections detected with DAB vs tyramide.
Sincerely,
George
p.s. Your current DAB precipitate IHC specimens might be suitable for
reflection (backscattered) confocal microscopy. DAB ppt's scatter
strongly in the blue (405 nm, 440 nm, 458 nm laser lines). You may need
to look up (or ask your vendor) your confocal microscope settings to get
a good backscatter signal. You can also visualize DAB on the confocal by
transmitted light (not a confocal mode). If you routinely counterstain
with hematoxylin, you can even acquire an RGB transmitted light image,
i.e. 458 nm for blue, 514 nm for green, 633 nm for red.
On 1/24/2011 5:27 AM, annaoru wrote:
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> 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!
> I´m a student doing my exam-project where we are using Arabidopsis Thaliana
> to express a HIV protein (p24) in order to creat an edible vaccine.
> Previously I have fixated and embedded (paraffin wax) the tissues to
> vizualise it in Light microscope (via IHC). Now I need to do the same
> procedure but try to analyze the tissue using confocal microscope. For that
> I need to know if I can use the same fixation solution for confocal as I did
> for Light microscope which was: egual volume of 0.2M Sodiumphosphate buffer
> pH 7.2 and Paraformaldehyde (8%) solution?
> And also what kind of embedding should I use for the confocal?
> Since I have a lot of material left from my previous work I´m hoping to be
> able to use it as it is for the confocal.
> Is this possible?
>
> Thank you for replying!
>
> /Anna
>
--
George McNamara, PhD
Analytical Imaging Core Facility
University of Miami
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