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In my fairly limited experience, any storage of fixed plant tissue with GFP/YFP results in substantial loss of fluorescence. I have only looked at fixed fluorescent proteins immediately after fixation and processing, but it's worth trying on your material to see if you can store for longer before imaging.
For teaching, I have mounted immunofluorescently stained plant cells and tissues in antifade agent (Mowiol) and kept in the freezer for up to 10 years - FITC retains its fluorescence (if student experiments fail, you just haul these out of the freezer to show them), but have never tried this with fluorescent proteins. You could try freezing the mounted tissues after the protocol below.
Here's a protocol for onion epidermal peels transiently-expressing fluorescent protein after DNA bombardment, from colleague David Collings - it's really for antibody labelling of FP-tagged tissue:
Use six-well tissue culture plates (large wells).
Float epidermal peels mesophyll-side downwards on PME solution (50 mM PIPES pH 7.0, 2 mM MgSO4, 2 mM EGTA) containing 1.0% (v/v) DMSO (1 min)
Fix in this solution containing 3.7% formaldehyde (1 h)
Wash with PME (2 x 10 min)
[Omit this section:
Digest cell walls with 1% (w/v) cellulysin Y6 and 0.1% (w/v) pectolyase Y23 (MP Biomedicals) in a PME solution containing 1.0% (w/v) bovine serum albumin and 0.4 M mannitol (2 min)
Wash with phosphate buffered saline (PBS; 131 mM NaCl, 5.1 mM Na2HPO4, 1.56 mM KH2PO4 pH 7.2) (2 x 5 min)
Block in incubation buffer (IB; PBS containing 1.0% BSA and 0.1% Tween-20) (10 min)
Incubate in primary antibodies (1 h, diluted in IB)
Washed in PBS (3 x 10 min)
Incubate in secondary antibodies (1 h, diluted in IB)
Washed in PBS (2 x 10 min)
Stain nuclei stained with DAPI (1 ėg.ml-1 in PBS, 5 min)]
Remove from the tissue culture plates by filling wells with [PBS] PME, slip coverslips (40 x 22 mm) beneath the floating peels and lift out.
Mount coverslips and peels on slides in AF1 antifade agent (Citifluor, London, England) and leave unsealed.
Good luck,
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
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From: Confocal Microscopy List [[log in to unmask]] on behalf of Jurkevic, Aleksandr [[log in to unmask]]
Sent: Friday, 30 August 2013 12:34 a.m.
To: [log in to unmask]
Subject: Leaf preservation after fixation for CFP and YFP imaging
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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/
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