9. Ethanol.
10. Histoclear.
11. Wax.
12. 58
C Oven.
13. Tissue embedding and processing cassettes.
14. Glass slides: Fisherbrand™ Tissue Path Superfrost™ Plus Gold
Slides.
15. Paintbrushes: #0 Watercolor paintbrushes.
16. Paraffin microtome.
17. Vacuum bell jar and vacuum pump.
2.2 Pre-hybridization
Slide Preparation
1. Ethanol series: Prepare a dilution series by adding water to
200 proof ethanol: 100%, 100%, 95%, 80%, 70%, 50%, 30%,
and 10%. Put at least 250 mL of each dilution into a glass
container.
2. Histo-Clear solution: Use 100% solution.
3. Protease stock solution: 50 mg/mL Protease. Dissolve 0.5 g
protease in 10 mL water. Predigest the solution by incubating
at 37
C for 4 h. Store 650 μL aliquots at À20
C.
4. TE solution: 10 mM Tris–HCl, 1 mM disodium EDTA,
pH 8.0.
5. TE-protease solution: Prepare right before use. Pre-warm TE
solution to 37
C. Mix 1 aliquot (650 μL) of 50 mg/mL
protease in 250 mL of TE solution.
6. 37
C Incubator.
7. Phosphate-buffered saline (PBS; 1Â): Dilute from PBS buffer
(10Â) with water.
8. 10% Glycine solution: Dissolve 5 g glycine in 50 mL water,
filter sterilize, and then store at 4
C.
9. 0.2% Glycine solution: Add 5 mL 10% glycine solution to
250 mL of PBS (1Â).
10. TAE buffer: To 393 mL of nuclease-free water, add 5.2 mL of
triethanolamine and 1.6 mL of HCl. Add 2 mL of acetic
anhydride right before use. TAE buffer should be prepared in
the chemical fume hood.
11. Glass staining dishes.
12. Slide holder (25 slide unit).
2.3 Hybridization
1. 100 mg/mL tRNA: Dilute 1 g tRNA in 1 mL RNase-free
water. Store at À20
C.
2. 50% Dextran sulfate solution: Add about 7 mL water to 5 g
dextran sulfate powder. Heat the solution at 80
C for about
Single-Molecule FISH Imaging of RNAs in Plants
25
10. Histoclear.
11. Wax.
12. 58
C Oven.
13. Tissue embedding and processing cassettes.
14. Glass slides: Fisherbrand™ Tissue Path Superfrost™ Plus Gold
Slides.
15. Paintbrushes: #0 Watercolor paintbrushes.
16. Paraffin microtome.
17. Vacuum bell jar and vacuum pump.
2.2 Pre-hybridization
Slide Preparation
1. Ethanol series: Prepare a dilution series by adding water to
200 proof ethanol: 100%, 100%, 95%, 80%, 70%, 50%, 30%,
and 10%. Put at least 250 mL of each dilution into a glass
container.
2. Histo-Clear solution: Use 100% solution.
3. Protease stock solution: 50 mg/mL Protease. Dissolve 0.5 g
protease in 10 mL water. Predigest the solution by incubating
at 37
C for 4 h. Store 650 μL aliquots at À20
C.
4. TE solution: 10 mM Tris–HCl, 1 mM disodium EDTA,
pH 8.0.
5. TE-protease solution: Prepare right before use. Pre-warm TE
solution to 37
C. Mix 1 aliquot (650 μL) of 50 mg/mL
protease in 250 mL of TE solution.
6. 37
C Incubator.
7. Phosphate-buffered saline (PBS; 1Â): Dilute from PBS buffer
(10Â) with water.
8. 10% Glycine solution: Dissolve 5 g glycine in 50 mL water,
filter sterilize, and then store at 4
C.
9. 0.2% Glycine solution: Add 5 mL 10% glycine solution to
250 mL of PBS (1Â).
10. TAE buffer: To 393 mL of nuclease-free water, add 5.2 mL of
triethanolamine and 1.6 mL of HCl. Add 2 mL of acetic
anhydride right before use. TAE buffer should be prepared in
the chemical fume hood.
11. Glass staining dishes.
12. Slide holder (25 slide unit).
2.3 Hybridization
1. 100 mg/mL tRNA: Dilute 1 g tRNA in 1 mL RNase-free
water. Store at À20
C.
2. 50% Dextran sulfate solution: Add about 7 mL water to 5 g
dextran sulfate powder. Heat the solution at 80
C for about
Single-Molecule FISH Imaging of RNAs in Plants
25