Leaf preservation after fixation for CFP and YFP imaging

[Jurkevic, Aleksandr]

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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:    [phone]
Fax:           [phone]
website  http://www.biotech.missouri.edu/mcc/

[Rosemary.White]

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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 μg.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
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Jurkevic, Aleksandr [[hidden email]]
Sent: Friday, 30 August 2013 12:34 a.m.
To: [hidden email]
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:    [phone]
Fax:           [phone]
website  http://www.biotech.missouri.edu/mcc/

[Stanislav Vitha-2]

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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
<[hidden email]> 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:    [phone]

>Fax:           [phone]

>website  http://www.biotech.missouri.edu/mcc/