Standard protocol for nuclei isolation
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tissue: root tips, rossette leaves (2-3weeks), flower buds etc.
1. fix in 2% (for root tips) or 4% (for leaves) formaldehyde/Tris buffer (Tris buffer: 10mM Tris, 10
mM Na2EDTA, 100 mm NaCl, 0.1% Triton X-100, pH 7.5), 20 min on ice, under vacuum
2. wash 2x10min in Tris buffer
3. in Petri dish chop the tissue with a razor blade in 250 μl of isolation buffer (15 mM Tris, 2 mM
Na2EDTA, 0.5 mM spermidin, 20 mM NaCl, 80 mM KCl, 15 mM mercaptoethanol, 0.1% Triton
X-100, pH 7.5), add more if necessary (for 4 small rosette leaves 500 μl is usually used)
4. homogenize the suspension briefly (20sec) by 11000rpm
5. filter through 32 μm mesh filter to remove tissue debries
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for flow-sorting of nuclei, the suspension of nuclei is further diluted with isolation buffer to 2 ml
and stained with DAPI, to final concentration 1 μg/ml, keep on ice
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flow-cytometric analysis of nuclei, setting the gates for sorting of 2C, 4C, 8C nuclei etc.
(technical details concerning the flow-sorter setup → Armin Meister)
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about 1000 nuclei of each sorted fraction are sorted directly onto a microscopic slide into a
drop of buffer containing 5% sucrose (100 mM Tris-HCl, 50 mM KCl, 2 mM MgCl2, 0.05%
Tween 20, 5% sucrose). The drop is let to air-dry at room temperature (the trick here is that
sucrose prevents complete air-drying, and thus the nuclei can be further used for
immunodetection of histones, or other proteins, but they could be used for detection of 5-mC,
or for fluorescent in situ hybridization as well). The slides can be used directly or stored at 4°C
for a short time (~days), or at –20°C up to several months. Repeated freezing and thawing of
the slides should be avoided.
Protocol for nuclei isolation (Paul Fransz)
1. chop the leaves with a sharp razor blade in 1 ml nuclei isolation buffer (NIB: 10 mM Tris-HCl,
pH 9.5, 10 mM EDTA, 100 mM KCl, 0.5 M sucrose, 4 mM spermidine, 1 mM spermine, 0.1%
(v/v) 2-mercaptoethanol) in a Petri dish on ice until a fine suspension
2. filter the suspension through a 20 μm mesh nylon sieve into an Eppendorf tube
3. fix the nuclei by adding an equal amount of 4% paraformadehyde and keep on ice for 30 min
4. centrifuge the suspension at 2500 rpm for 4 min at 4°C
5. resuspend the pellet in about 50 μl NIB
6. pipet 3 μl onto a clean slide
7. let the droplet air-dry
Histone Immunolabeling – general protocol
1. postfix the slides in 4% paraformaldehyde/PBS (freshly prepared), 15-20min (on ice)
2. was 3x5 min in PBS
3. blocking – at 37°C in a humid chamber, 30 min – 1h, 30-100 μl/slide, cover with cover slip
(blocking buffer: PBS + 3% BSA, 10% horse serum)
4. incubation with primary antisera: Upstate antibodies overnight at 4°C in a humid chamber,
some other work also when incubated at RT or 37°C, 30 min – 1h (incubation buffer: PBS +
1% BSA, 10% horse serum, 0.1% Tween 20)
5. wash 3x5min (or longer) in PBS
6. incubation with secondary antibodies: at 37°C, 30min – 1h
7. wash 3x5min (or longer) in PBS (avoid direct light!)
8. dehydrate the slides through ethanol series: 70, 90, 96% ethanol, 2 min each, and let to airdry (avoid direct light)
9. apply DNA counterstaining: DAPI in Vectashield (1-2 μg/ml) 8-12 μl, depending on the size of
cover slip (22x22 or 24-32 mm, respectively)
notes:
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try to prevent any drying-out of slides during the procedure, it might introduce artefacts and
increase background
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for blocking and incubation with antibodies, alternatively PBS with BSA (2-8%) and Tween 20
(0.05-0.1%) as a detergent (helping antibodies to penetrate into the material) could be used,
volume depends on the area occupied by the nuclei
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instead of cover slips the cut pieces of Parafilm could be used for blocking and incubations
steps
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washing step between blocking and incub. with prim. antisera is not necessary, just shake off
the excess of blocking buffer and apply the drop of diluted primary antisera
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the dilutions of primary and secondary (or tertiary, when necessary) antibodies must be tested
individually
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gentle shaking during washing steps might help but is not necessary in case of isolated nuclei
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step 8 can be omitted (depends on a personal preference), in this case the excess of PBS is
shaken off the slides and the drop of 5 μl of DAPI diluted in Vectashield (volume for 22x22 mm
coverslip) is applied
Fluorescent In situ Hybridization after Immunolabeling
For the combined detection of H3-dimethylK9 and 5S rDNA, immunodetection of histones is done first.
After evaluation and capturing the images, the slides are processed for subsequent FISH.
1. Label DNA according to manufacturer’s protocol (Roche, DIG-Nick translation Mix or
Biotin-Nick Translation Mix). Store at -20°C.
2. Wash the slides in 4X SSC (1X SSC is 0.15 M NaCl and 0.015 M sodium citrate) plus
0.1% Tween 20 to remove the cover slip, and then wash briefly in 2X SSC.
3. Postfix the nuclei in 4% paraformaldehyde/2 x SSC, wash in 2X SSC, dehydrate in 70 and
96% ethanol, and air-dry.
4. Prepare and denature 20 µl of the hybridization mixture containing the probe (150-300
ng), 50% formamide, 10% Dextran Sulfate and 2X SSC, at 80°C for 2 min. Let cool on ice.
5. Add the hybridization mixture to the slide and cover with a 22x22 mm cover slip.
6. Denature target DNA by heating the slides on a hot block at 80°C for 2 min.
7. Incubate in a moist chamber overnight at 37°C.
8. Wash the slides in jars containing SF50 (50% formamide, 2X SSC, pH 7) at 42°C for 3x10
min. Wash briefly in 2X SSC.
Immunodetection of 2 different probes labelled with Biotin and Digoxigenin: Adapt the procedure to
your probe(s)
1. Rinse the slides briefly in 4T (4X SSC with 0.05% (v/v) Tween-20).
2. Incubate in 4M (4X SSC with 5% non-fat dry milk) at 37°C for 30 min in a moist chamber,
rinse briefly in 4T.
3. Incubate in 5 µg/ml Avidin~Texas Red (Vector laboratories) in 4M in a moist chamber at
37°C for 30 min. Rinse in 4T for 2x5 min.
4. Rinse in TNT (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween-20) for 5 min.
5. Incubate in 5 µg/ml goat-anti-avidin~biotin (Vector laboratories)and 0.2 µg/ml mouse-antidigoxigenin (Roche) in TNB (100 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Roche
Blocking reagent) in a moist chamber at 37°C for 30 min.
6. Rinse in TNT for 3x5 min.
7. Incubate in 5 µg/ml Avidin~Texas red and 1:1000 diluted rabbit-anti-mouse~FITC (Sigma)
in TNB in a moist chamber at 37°C for 30 min.
8. Rinse in TNT for 3x5 min.
9. Incubate in 1:200 diluted Alexa 488-conjugated goat-anti-rabbit (Molecular Probes) in TNB
in a moist chamber at 37°C for 30 min.
10. Rinse in TNT for 3x5 min.
11. Dehydrate in 70 and 96% ethanol, 1 min each and air-dry.
12. Mount in Vectashield (Vector laboratories) with DAPI (2 µg/ml).