*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
*****
For Arabidopsis leaf tissue, I take the leaf segments, vacuum infiltrate with
the fixative (~3% formaldehyde freshly prepared from paraformaldehyde,
in 50 mM phosphate buffer pH 7, or in PBS), and let sit for 30 min. I then
apply microwave fixation (total 6 min, consisting of 2 min ON, 2 min OFF,
2 min ON, using Pelco Biowave with the ColdSpot chiller set to 20 C, power
set to 250W, vacuum cycling on/off every 30 seconds). It appears that
microwave fixation preserves the fluorescent protein signal better than just
leaving the tissue in the fixative on the bench.
After the microwave fixation, I rinse in the same buffer several times, with
1 min microwave irradiation in every rinse to accelerate the process (the
same microwave settings as given above).
Then I infiltrate the tissue with a glycerol based mounting medium
(essentially ~80% glycerol buffered to pH 8.5, with n-propyl gallate as
antifade; recipe from Applied Precision handout is below). I leave the
samples in a refrigerator overnight, then store them in -20C until needed.
I normally do not worry about tissue shrinkage since my interest is in sub-
cellular details rather than whole-tissue structure.
nPG (n-propyl gallate) Antifade Mounting Media Preparation:
1. In a 50 ml falcon tube, add:
5ml of 0.2M TRIS, pH 8.5
43 ml glycerol (check for autofluorescence first!)
2.5g n-propyl gallate
2. Wrap tube completely in foil to protect from light
3. Mix on stirrer until dissolved (overnight)
Stan Vitha
Texas A&M University
Microscopy and Imaging Center
On Thu, 29 Aug 2013 14:34:12 +0000, Jurkevic, Aleksandr
<[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
>*****
>
>Hello,
>
>One of our clients needs to preserve a large amount of fixed leaf samples
(leaf disks) expressing YFP and CFP tags. He wants to fix the samples with
buffered 4% PFA for a few hours and then store them in the cryoprotective
solution (e.g., Watson RE et al., Peptides 1986;7:155-159) at -20C until
imaging (within 2-3 weeks after fixation). I wonder whether anybody from
the plant researcher community has tried this approach and whether it
helped to retain CFP or YFP fluorescence at reasonable levels. Are there
any other good options? Thank you.
>
>Alexander
>
>
>Alexander Jurkevic, PhD
>Associate Director
>Molecular Cytology Core
>University of Missouri
>120 Life Sciences Center
>1201 E. Rollins St.
>Columbia, MO 65211-7310
>
>Phone: 573-882-4895
>Fax: 573-884-9676
>website http://www.biotech.missouri.edu/mcc/
|
