6. Leave the syringe for 15 min in the caulking gun to ensure that
residual air is replaced by buffer solution. Repeat if necessary.
7. Place degassed leaf sample ventral side up (position of cutoff
corner on the left upper side) on a glass slide.
8. Add a droplet of buffer solution before covering the sample
with a 40 Â 24 mm coverslip.
9. Fix the short ends of the coverslip with sticking tape.
10. Remove air inclusions between sample and coverslip by careful
tapping with the blunt end of a tweezer.
11. Fill empty space between coverslip and glass slide with buffer,
and then seal the long ends with fast drying and easily removable fixogum.
3.3.2 Analysis of
Telomeric GFP Signals
1. Place the slide in the microscope and examine with a 40Â NA
1.2 water objective.
2. Select appropriate locations without air inclusions since the
latter interfere with signal recording.
3. Select optimal resolution settings to avoid over- or
undersampling.
4. GFP is excited with a 488 nm laser line and emission is detected
over a range of 490–540 nm.
5. Collect telomeric GFP distribution as Z-stacks.
3.4 Combination of
Immunostaining and
FISH for Confirming
the Specificity of
CRISPR/dCas9-Caused
Signals
1. Cut a 1 cm
3 piece of leaf from the infiltrated area, transfer into
4% paraformaldehyde solution, and fix on ice for 5 min under
vacuum followed by 30 min under atmospheric pressure (see
Note 13).
2. Remove the paraformaldehyde solution and wash twice in PBS.
3. In a drop of LB01 lysis buffer in a petri dish, finely chop the leaf
using a sharp razor blade (see Note 14).
4. Add 500 μL LB01 lysis buffer to the homogenate and transfer
into 35 μm cell suspension filter tube.
5. Apply 200 μL of the homogenate to a cytology funnel and
centrifuge for 5 min at 450 rpm in a cytocentrifuge (see
Note 2).
6. Wash slides two times in PBS for 5 min and add 60 μL of 4%
BSA followed by incubation at room temperature for 45 min in
a high-humidity plastic box. Carefully cover slides with
parafilm tape.
7. Wash slides two times in PBS for 5 min and add 60 μL of GFP
antibody solution (2% (vol/vol) BSA in PBS, GFP antibody in
1:2500 dilution) followed by incubation at room temperature
for 1 h in a high-humidity plastic box. Carefully cover slides
with parafilm tape.
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Solmaz Khosravi et al.
residual air is replaced by buffer solution. Repeat if necessary.
7. Place degassed leaf sample ventral side up (position of cutoff
corner on the left upper side) on a glass slide.
8. Add a droplet of buffer solution before covering the sample
with a 40 Â 24 mm coverslip.
9. Fix the short ends of the coverslip with sticking tape.
10. Remove air inclusions between sample and coverslip by careful
tapping with the blunt end of a tweezer.
11. Fill empty space between coverslip and glass slide with buffer,
and then seal the long ends with fast drying and easily removable fixogum.
3.3.2 Analysis of
Telomeric GFP Signals
1. Place the slide in the microscope and examine with a 40Â NA
1.2 water objective.
2. Select appropriate locations without air inclusions since the
latter interfere with signal recording.
3. Select optimal resolution settings to avoid over- or
undersampling.
4. GFP is excited with a 488 nm laser line and emission is detected
over a range of 490–540 nm.
5. Collect telomeric GFP distribution as Z-stacks.
3.4 Combination of
Immunostaining and
FISH for Confirming
the Specificity of
CRISPR/dCas9-Caused
Signals
1. Cut a 1 cm
3 piece of leaf from the infiltrated area, transfer into
4% paraformaldehyde solution, and fix on ice for 5 min under
vacuum followed by 30 min under atmospheric pressure (see
Note 13).
2. Remove the paraformaldehyde solution and wash twice in PBS.
3. In a drop of LB01 lysis buffer in a petri dish, finely chop the leaf
using a sharp razor blade (see Note 14).
4. Add 500 μL LB01 lysis buffer to the homogenate and transfer
into 35 μm cell suspension filter tube.
5. Apply 200 μL of the homogenate to a cytology funnel and
centrifuge for 5 min at 450 rpm in a cytocentrifuge (see
Note 2).
6. Wash slides two times in PBS for 5 min and add 60 μL of 4%
BSA followed by incubation at room temperature for 45 min in
a high-humidity plastic box. Carefully cover slides with
parafilm tape.
7. Wash slides two times in PBS for 5 min and add 60 μL of GFP
antibody solution (2% (vol/vol) BSA in PBS, GFP antibody in
1:2500 dilution) followed by incubation at room temperature
for 1 h in a high-humidity plastic box. Carefully cover slides
with parafilm tape.
352
Solmaz Khosravi et al.