6. Glutaraldehyde (GA).
7. Fixative solution (PFA, 0.1 M phosphate buffer (4%), GA
(0.05%), pH to 7.2).
8. Desiccator.
9. Ethanol, different concentrations (10, 20, 30, 50, 70, 80,
90, 95, 99.5%).
10. TopVision low melting point (LMP) agarose.
11. London Resin (LR) white resin medium grade, catalyzed.
12. Embedding capsules, 8 mm flat, polypropylene.
13. Vortex mixer, rotary or orbital shaker.
14. Incubator 60
C.
15. Ultramicrotome.
16. Thin brush.
17. Adhesion slides, Polysine
18. Toluidine blue stain for microscopy.
19. Slide warming plate.
20. Compressed air duster.
21. Light microscope.
2.3 AFM Hardware
and Cantilevers
1. Catalyst Bioscope with the optical microscope and NanoScope
V controller or bioscope catalyst AFM with the inverted Nicon
microscope (Bruker Nano Surfaces, Santa Barbara, CA)
combined.
2. Cantilevers with conical shape tips (tip radius 2–10 nm) and
spring constant in the range 0.3–0.7 N/m.
3. Sapphire disk pre-mounted on a 12 mm magnetic sample disk.
4. Tweezers with nonmetallic (plastic) tips.
2.4 Software
1. NanoScope 8.15 software to operate AFM.
2. NanoScope Analysis 150 software to analyze AFM data.
3. Computer (e.g., Intel 2.4 GHz Quad Core, 4 GB RAM, 500
GB hard drive) with Windows XP software.
3 Methods
3.1 Plant Material
Cultivation and
Fixation
1. Sterilize the seeds in a mixture of 70% ethanol with 0.1%
Tween-20 for 2 min, replaced by 95% ethanol for 1 min. Wait
for the seeds to dry.
2. Prepare sterile agar plates containing 1/2 MS, 1% sucrose, at
pH 5.6, with 0.7% plant agar.
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
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7. Fixative solution (PFA, 0.1 M phosphate buffer (4%), GA
(0.05%), pH to 7.2).
8. Desiccator.
9. Ethanol, different concentrations (10, 20, 30, 50, 70, 80,
90, 95, 99.5%).
10. TopVision low melting point (LMP) agarose.
11. London Resin (LR) white resin medium grade, catalyzed.
12. Embedding capsules, 8 mm flat, polypropylene.
13. Vortex mixer, rotary or orbital shaker.
14. Incubator 60
C.
15. Ultramicrotome.
16. Thin brush.
17. Adhesion slides, Polysine
18. Toluidine blue stain for microscopy.
19. Slide warming plate.
20. Compressed air duster.
21. Light microscope.
2.3 AFM Hardware
and Cantilevers
1. Catalyst Bioscope with the optical microscope and NanoScope
V controller or bioscope catalyst AFM with the inverted Nicon
microscope (Bruker Nano Surfaces, Santa Barbara, CA)
combined.
2. Cantilevers with conical shape tips (tip radius 2–10 nm) and
spring constant in the range 0.3–0.7 N/m.
3. Sapphire disk pre-mounted on a 12 mm magnetic sample disk.
4. Tweezers with nonmetallic (plastic) tips.
2.4 Software
1. NanoScope 8.15 software to operate AFM.
2. NanoScope Analysis 150 software to analyze AFM data.
3. Computer (e.g., Intel 2.4 GHz Quad Core, 4 GB RAM, 500
GB hard drive) with Windows XP software.
3 Methods
3.1 Plant Material
Cultivation and
Fixation
1. Sterilize the seeds in a mixture of 70% ethanol with 0.1%
Tween-20 for 2 min, replaced by 95% ethanol for 1 min. Wait
for the seeds to dry.
2. Prepare sterile agar plates containing 1/2 MS, 1% sucrose, at
pH 5.6, with 0.7% plant agar.
Atomic Force Microscopy to Study Cell Wall Mechanics in Plants
355