3. Tubes and racks for sample preparation.
4. Microcentrifuge.
5. Nickel grids for TEM samples.
6. Microtome.
7. Glass or diamond knife.
8. Transmission electron microscope (TEM) Hitachi H7000,
equipped with a SIS Megaview III digital camera.
3 Methods
3.1 Sample Fixation,
Preparation
and Inclusion in Resin
1. The fruit tissue sample should be cut into small squares sections
3–5 mm long and 1 mm wide and fixed in the main fixative
solution (3% glutaraldehyde +4% formaldehyde in 0.1 M
PIPES buffer at pH 7.2). The tissue sample should be fixed
for at least 3 h. Be aware of a fast inclusion in the fixative in
order to avoid long contact with oxygen.
2. Rinse the sample with buffer (PIPES 0.1 M) for 10 min.
3. Postfix in buffered 1% osmium tetroxide prepared in 0.1 M
PIPES buffer. This step should not exceed more than 1 hour.
The sample could not become black in color. Move/rotate the
sample during the treatment.
4. Rinse the section with buffer (PIPES 0.1 M) 10 min.
5. Rinse the section briefly in water.
6. Place the fruit section in 2% uranyl acetate for 20 min (not
longer than 30 min) and move/rotate the sections during the
treatment. Uranyl acetate staining could be done at this step or
f
afterward (see Subheading 3.3).
7. Place the tissue for dehydration sequentially in the following
solutions: ethanol 30% for 10 min, ethanol 50% for 10 min,
ethanol 70% for 10 min, ethanol 95% for 10 min, absolute
ethanol for 20 min.
8. Place the section on acetonitrile for 10 min.
9. Place the section in 1 mL acetonitrile–Spurr resin (50:50)
overnight.
10. Place the fruit section in the spur resin solution in continuous
rotation during 6 h.
11. Embedding the fruit section in fresh epoxy resin (Spurr) allowing to polymerize in oven at 60
C for a period between 20 and
24 h.
12. Collect the rising blocks and store them property labelled in
dry environment.
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Joanna Lado et al.
4. Microcentrifuge.
5. Nickel grids for TEM samples.
6. Microtome.
7. Glass or diamond knife.
8. Transmission electron microscope (TEM) Hitachi H7000,
equipped with a SIS Megaview III digital camera.
3 Methods
3.1 Sample Fixation,
Preparation
and Inclusion in Resin
1. The fruit tissue sample should be cut into small squares sections
3–5 mm long and 1 mm wide and fixed in the main fixative
solution (3% glutaraldehyde +4% formaldehyde in 0.1 M
PIPES buffer at pH 7.2). The tissue sample should be fixed
for at least 3 h. Be aware of a fast inclusion in the fixative in
order to avoid long contact with oxygen.
2. Rinse the sample with buffer (PIPES 0.1 M) for 10 min.
3. Postfix in buffered 1% osmium tetroxide prepared in 0.1 M
PIPES buffer. This step should not exceed more than 1 hour.
The sample could not become black in color. Move/rotate the
sample during the treatment.
4. Rinse the section with buffer (PIPES 0.1 M) 10 min.
5. Rinse the section briefly in water.
6. Place the fruit section in 2% uranyl acetate for 20 min (not
longer than 30 min) and move/rotate the sections during the
treatment. Uranyl acetate staining could be done at this step or
f
afterward (see Subheading 3.3).
7. Place the tissue for dehydration sequentially in the following
solutions: ethanol 30% for 10 min, ethanol 50% for 10 min,
ethanol 70% for 10 min, ethanol 95% for 10 min, absolute
ethanol for 20 min.
8. Place the section on acetonitrile for 10 min.
9. Place the section in 1 mL acetonitrile–Spurr resin (50:50)
overnight.
10. Place the fruit section in the spur resin solution in continuous
rotation during 6 h.
11. Embedding the fruit section in fresh epoxy resin (Spurr) allowing to polymerize in oven at 60
C for a period between 20 and
24 h.
12. Collect the rising blocks and store them property labelled in
dry environment.
240
Joanna Lado et al.