Generation of Arabidopsis Leaf Nuclei for Protein Immunolocalization or DNA Fish (Pikaard lab October 2010)

Introduction: 

Protocol of Olga Pontes (Pikaard Laboratory, Washington University in St. Louis)

REPRESENTATIVE PUBLICATIONS:

Pontes, Olga, Carey Fei Li, Pedro Costa Nunes, Jeremy Haag, Thomas Ream, Alexa
Vitins, Steven E. Jacobsen and Craig S. Pikaard (2006). The Arabidopsis chromatinmodifying
nuclear siRNA pathway involves a nucleolar RNA processing center. Cell
126: 79-92.

Richard J. Lawrence, Keith Earley, Olga Pontes, Manuela Silva, Z. Jeffrey Chen, Nuno
Neves, Wanda Viegas and Craig S. Pikaard (2004). A concerted DNA
methylation/histone methylation switch regulates rRNA gene dosage control and
nucleolar dominance. Molecular Cell 13: 599-609.

Pontes, Olga, Richard J. Lawrence, Nuno Neves, Manuela Silva, Jae-Hyeok Lee, Z.
Jeffrey Chen, Wanda Viegas, and Craig S. Pikaard (2003). Natural variation in nucleolar
dominance reveals the relationship between nucleolus organizer chromatin topology and
rRNA gene transcription in Arabidopsis. Proc. Natl. Acad. Sci. USA 100:11418-11423.

Pontes, Olga, Nuno Neves, Manuela Silva, Michelle S. Lewis, Andreas Madlung, Luca
Comai, Wanda Viegas and Craig S. Pikaard (2004). Chromosomal locus rearrangements
are a rapid response to formation of the allotetraploid Arabidopsis suecica genome. Proc.
Natl. Acad. Sci. USA 101: 18240-18245.

Procedure : 

Materials and Reagents

-Nuclear extraction buffer (NEB)
10 mM Tris-HCl pH 9.5
10 mM KCl
0.5 M sucrose
4 mM spermidine
10 mM spermine
0.1% 2-mercaptoethanol
- 8% formaldehyde (or paraformaldehyde). Formaldehyde comes as a 37% stock
(Sigma), so one dilutes ~4.5 fold to make 8% formaldehyde.
- Nylon mesh with mesh sizes of 20 (optional), 50- and 100 μm.
-Plastic petri dishes
-razor blades
-Glass slides (we use Fisherbrand frosted slides) and coverslips
- DAPI (4'-6-Diamidino-2-phenylindole) in CITIFLUOR antifade solution (1μg/μl)
purchased from Electron Microscopy Sciences, Hatfield , PA

Preparation of nuclei

- In the lid of a plastic petri dish, lying on a bed of ice, Chop 1g of fresh leaf tissue from
~3 week old plants grown in soil. Rosettes of these plants are ~ 3 cm in diameter.
Choose intermediate-sized leaves (neither the oldest nor youngest) for protein
immunolocalization, but choose smaller, newly emerged leaves for RNA FISH detection
of siRNAs). Chop the leaves in 1-2 ml of NEB using a razor blade until you get as fine a
suspension as possible. This will take 5 minutes or more of vigorous mincing. Keep in
mind that you are trying to chop the tissue sufficiently that nuclei are released from
disrupted cells, so the tissue must be chopped extremely fine.
-Using a blue pipette tip, with its tip cut off to make the bore wider, pipette the
homogenate from the lid of the petri dish into a 15 ml conical tube, on ice.
- add a volume of 8% formaldehyde that is equal to the volume of NEB used for
homogenation. Mix by swirling and allow nuclei/tissue to fix for 20 min, on ice.
-Meanwhile, make filters from blue pipette tips and nylon mesh of 50 and 100μm:
Cut ~3 cm off the tips of blue pipette tips, where the bore is not yet full width,
such that the cut tip can fit most of the way into a 1.5 ml microcentrifuge tube. Place the
cut tip near a blue flame, while twirling, for 1-2 seconds to melt the plastic slightly and
while still molten, puch the tip down onto a sheet of 50 or 100 μm nylon mesh so that the
mesh adheres to the tip. Trim off excess mesh material. The filter tip will now just fit into
the top of a 1.5 ml microcentrifuge tube. Make both 50 and 100 μm filters by this
method.
- Filter the homogenate first through the 100 μm filter and then through the 50 μm filter.
- Centrifuge the homogenate 3 min at only 2.5rpm at 4ºC. You should see a
whitish/grayish smudge (the nuclei!) at the center of a green pellet
- Resuspend the pellet in 40μl of NEB. The white/gray smudge will be the hardest part to
resuspend.
- Apply 3 μl onto individual slides, near the end of the slide furthest from the frosted
writing surface. Don't go over the same area twice, but go back and forth in adjacent
parallel passes to achieve a thin film covering a 2-3 cm square. Slides will dry quickly in
the air.
- Check the quality of the nuclei by adding a drop of DAPI (in CITIFLUOR). Cover with
a coverslip and store in the dark for several hours. Cardboard slide storage books are
useful for this step.
- Slides can be stored at 4ºC for up to one month.

IMMUNOSTAINING OF CHROMOSOMAL PROTEINS IN PLANT CELLS

Solutions and reagents

- 4% PFA (paraformaldehyde) or formaldehyde
- 10X KPBS
1.28M NaCl
20mM KCl
80mM Na2HPO4
20mM KH2PO4 (pH 7.2)
- DAPI at 1μg/μl in CITIFLUOR
- Triton X-100
- Blocking Solution: 1% BSA Fraction V in KPBS 0.1% Triton X
- Primary and secondary antibodies, typically 1:200 to 1:1000 dilutions in Blocking
Solution.

Primary antibody incubation

- (Optional) re-fix preparations in formaldehyde 30 min and wash 3X 5min in
KPBS/0.1% Triton X.
- Block preparation by placing 200 μl blocking solution onto the slide and cover with a
22x22 mm coverslip. Incubate in a humid chamber for 30 min at 37ºC.
- Wash off blocking solution 3X 5min in KPBS/0.1% Triton X.
- Add primary antibody solution (50-100 μl per slide) in blocking solution. Cover with a
22x22 mm coverslip and place in the humid chamber overnight at 4ºC.

Detection, Counterstaining and Mounting

- Wash 3X 5 min in KPBS.
- Block with blocking solution 30 min, in a humid chamber for 30 min at 37ºC.
- Add the proper secondary antibody diluted in KPBS/0.1% Triton X, cover with 22x22
mm coverslip and incubate 2h at 37ºC.
- Wash 3X 10 min in KPBS and mount the slides in DAPI and cover with a coverslip
22x40 mm. Keep at 4ºC.

FLUORESCENT IN SITU HIBRIDIZATION (FISH)

Solutions and reagents

- 10 mM HCL
- Pepsin
10 mg/ ml in 10 mM HCL
- RNAse A
100μg/ml in 10 mM Tris.HCl, pH 8.0)
- Tween20
- 20X SSC
3M NaCl
0.3M Sodium Citrate (pH 7.5).
- 4X SSC/0.2% Tween-20
- 2X SSC
- Formamide
- Dextran sulfate
- SDS (sodium dodecyl sulfate)
- Salmon sperm
- Blocking solution: 1% BSA Fraction V in 4X SSC/0.2% Tween20
- Detection solution: Digoxigenin (Fab fragments) conjugated to FITC (dilution 1:50 of
200 μg/ml) for digoxigenin labeled probes; and Cy3-Streptavin (1:500 of 1 mg/ml) in the
blocking solution.
- DAPI at 1μg/μl in CITIFLUOR antifade solution

DNA probe labeling

Nick Translation (Roche Kit), according to the manufacturer’s description, or by PCR.

Pretreatment of chromosome preparations

- Incubate the preparations for 10 min at 37°C, in a humid chamber, with 200 μl pepsin
for each slide. Cover with a cover slip (22x22 mm), and wash 3X in 2xSSC.
- Add 200μl of RNAse A to each slide. Cover with a cover slip (22x22 mm), incubate in
a humid chamber for 1h at 37°C, and then wash 3X in 2XSSC.
- Post-fix 10 min at room temperature in 4% formaldehyde. After this treatment wash the
slides 3X in 2X SSC.
- Dehydrate slides through a 70%, 90% and 100% ethanol series, 3 min each, and air-dry.

In situ hybridization mixture and hybridization conditions

The hybridization mixture is prepared according to optimized conditions, found
empirically. The stringency at which an in situ experiment is carried out reflects the
approximate percentage of nucleotides that are correctly matched in the probe and the
target. Stringency depends on the GC content, length of the probe, salt concentration,
formamide concentration and the reaction temperature. The hybridization mixture
contains sonicated salmon sperm, dextran sulfate and SDS (sodium dodecyl sulfate). A
typical hybridization mixture is exemplified in the table I.

Table I. In situ hybridization mixture. Components and concentrations

Stock solution Final concentration in mixture
100% formamide 50%
20X SSC 2X
50% dextran sulfate (w/v) 10%
10% SDS (w/v) 0.1%
Salmon sperm (10 μg/ml) 1.5 μg/μl
Probe 100-250 ng each
Sterilize water Adjusted to a final volume of 20-30μl
- Add the hybridization mixture and cover with a coverslip 22x22 mm coverslip.
Denature at 80°C for 5 min on a modified thermocycler (thermocycler JMResearch PCT-
100). Place the slides overnight in a humid chamber at 37°C; make sure the chamber is
pre-warmed.

Post-hybridization washes and detection of hybridization sites

- Post hybridization washes are carried out using 20 % (v/v) formamide in 0.1X SSC at
42°C for 10 min.
-Wash the slides 2x 5min in 2X SSC at 42°C, and 2x in 4X SSC/0.2% Tween-20 at room
temperature.
- Block the slides for 15 min at room temperature with blocking solution.
- The detection solution contains anti-digoxigenin (Fab fragments) conjugated to FITC or
Cy3-Streptavin and the incubation is performed in humid chamber for 1h at 37°C.
- Wash the slides in 4x SSC/0.2% Tween-20 for 3x 5 min.
- Mount the slides in a drop of DAPI and cover with coverslip 22x40 mm. Keep at 4ºC.

National Science Foundation Logo

The Epigenomics of Plants
International Consortium
Web site is made possible
by a grant from the

National Science Foundation