3.2 Sample Fixation
1. Dissect the samples (maize anthers as an example in this protocol) using a dissecting microscope with a fine knife and forceps.
2. Place samples in 20 mL glass scintillation vials. Immediately
immerse the sample in fixation buffer. The volume of fixation
buffer should be at least 15 times the relative volume of the
sample.
3. Apply 0.08 MPa vacuum to the sample in a bell jar for 15 min
and then release the pressure. Repeat this step three times or
until the samples sink (see Note 2).
4. Store the samples at 4
C. Samples can be stored for up to
7 days.
3.3 Paraffin
Embedding
and Sectioning
1. Fixed samples are then rinsed with PBS (1Â) buffer for 30 min.
And then dehydrate by going through an ethanol series
(0%, 10%, 30%, 50%, 70%, 90%, 100%), 30 min each step.
2. Dehydrate the sample an additional two times, 30 min each
time with 100% ethanol. Then leave the sample in 100% ethanol at 4
C overnight.
3. Warm up the sample to room temperature.
4. Treat the sample with ethanol/Histo-Clear series (75% ethanol/25% Histo-Clear, 50% ethanol/50% Histo-Clear, 25% ethanol/75% Histo-Clear) for 1 h each step.
5. Immerse the sample in pure Histo-Clear three times, 1 h
each time.
6. Leave about 5 mL Histo-Clear in the glass scintillation vial, add
wax to fill the vial, and leave it in a 58
C oven overnight.
7. Add wax to the vial every hour till the vial is completely filled
with melted wax. Pour out the solution, and add in pure
melted wax.
8. Replace the wax every 3 h four times.
9. Cast the processed samples onto the tissue-embedding and
-processing cassettes and wait till the wax is solidified and
completely cool.
10. Paraffin-embedded samples can be stored at 4
C for up to
6 months.
11. Start a 50
C water bath and 37
C slide warmer.
12. Trim paraffin blocks into a trapezoid shape with a thin layer
(about 2–3 mm) of paraffin on each side of the sample.
13. Collect sections with about 10 μm thickness. Examine the
sections using a dissecting microscope to make sure that the
collected sections have the desired morphology. Transfer 2–4
sections to the water bath with a paintbrush. Collect the sections onto a glass slide.
14. Leave the slides on the slide warmer to dry for at least 24 h.
Dried slides can be stored at 4
C for up to 1 week.
Single-Molecule FISH Imaging of RNAs in Plants
27
1. Dissect the samples (maize anthers as an example in this protocol) using a dissecting microscope with a fine knife and forceps.
2. Place samples in 20 mL glass scintillation vials. Immediately
immerse the sample in fixation buffer. The volume of fixation
buffer should be at least 15 times the relative volume of the
sample.
3. Apply 0.08 MPa vacuum to the sample in a bell jar for 15 min
and then release the pressure. Repeat this step three times or
until the samples sink (see Note 2).
4. Store the samples at 4
C. Samples can be stored for up to
7 days.
3.3 Paraffin
Embedding
and Sectioning
1. Fixed samples are then rinsed with PBS (1Â) buffer for 30 min.
And then dehydrate by going through an ethanol series
(0%, 10%, 30%, 50%, 70%, 90%, 100%), 30 min each step.
2. Dehydrate the sample an additional two times, 30 min each
time with 100% ethanol. Then leave the sample in 100% ethanol at 4
C overnight.
3. Warm up the sample to room temperature.
4. Treat the sample with ethanol/Histo-Clear series (75% ethanol/25% Histo-Clear, 50% ethanol/50% Histo-Clear, 25% ethanol/75% Histo-Clear) for 1 h each step.
5. Immerse the sample in pure Histo-Clear three times, 1 h
each time.
6. Leave about 5 mL Histo-Clear in the glass scintillation vial, add
wax to fill the vial, and leave it in a 58
C oven overnight.
7. Add wax to the vial every hour till the vial is completely filled
with melted wax. Pour out the solution, and add in pure
melted wax.
8. Replace the wax every 3 h four times.
9. Cast the processed samples onto the tissue-embedding and
-processing cassettes and wait till the wax is solidified and
completely cool.
10. Paraffin-embedded samples can be stored at 4
C for up to
6 months.
11. Start a 50
C water bath and 37
C slide warmer.
12. Trim paraffin blocks into a trapezoid shape with a thin layer
(about 2–3 mm) of paraffin on each side of the sample.
13. Collect sections with about 10 μm thickness. Examine the
sections using a dissecting microscope to make sure that the
collected sections have the desired morphology. Transfer 2–4
sections to the water bath with a paintbrush. Collect the sections onto a glass slide.
14. Leave the slides on the slide warmer to dry for at least 24 h.
Dried slides can be stored at 4
C for up to 1 week.
Single-Molecule FISH Imaging of RNAs in Plants
27