3.2 Cutting
Procedure
A microtome should be used; a diamond knife is highly recommended, but a glass knife could be also useful, which may be
replaced after 2 or 3 cuttings.
1. Place the sample in the correct orientation.
2. Prepare and cut the resin end with a knife or scalpel.
3. Put water in the knife collector/recipient in order to allow the
thin sections to launch on the water surface and to be easily
recovered.
4. The thin sections could be first observed by optic microscopy
in order to guarantee preservation of the tissue structure and
quality.
5. A chloroform drop should be included in the water for section
stretching before picking.
6. Fish sections with an instrument containing the selected grid.
Tissue to be observed should be localized in the center of the
grid. Sections should be put in nickel grids (and not copper).
7. Leave the sections to dry for 24 h before staining.
3.3 Grid Staining
1. Place a piece of wax-paper into a petri dish.
2. Invert grids section side down onto drops of uranyl acetate
solution for 15 min in the dark (see Notes 5 and 6).
3. Wash gently in a stream of distilled water and blot dry.
4. Invert grids section side down onto drops of Reynolds lead
stain (5 min) in the presence of NaOH pellets (in order to
absorb CO 2 ). A petri dish with cover is a suitable place to avoid
gas exchange. Lead stain produces a precipitate on exposure to
carbon dioxide. Be careful and avoid breathing into samples
during staining (see Note 7).
5. Wash gently in a stream of distilled water and blot dry (see
Notes 8 and 9).
6. Store the staining grids in appropriate racks, properly identified, until microscopic observation (see Note 9).
3.4 TEM
Observations
1. To initiate TEM observation of the different grids, the microscope should be property adjusted following the Microscopy
Unit instructions and working protocols that may vary for each
particular microscope.
2. As a general procedure, the grid should be mounted over the
sample holder, properly adjusted to the microscope allowing
then perform the vacuum.
3. Selection of appropriate voltage and brightness, and searching
for observation cell sections of interest usually at low magnifications to get a wider image of the sections. Increase
Structure of Plastid-Containing Carotenoids from Fruits
241
Procedure
A microtome should be used; a diamond knife is highly recommended, but a glass knife could be also useful, which may be
replaced after 2 or 3 cuttings.
1. Place the sample in the correct orientation.
2. Prepare and cut the resin end with a knife or scalpel.
3. Put water in the knife collector/recipient in order to allow the
thin sections to launch on the water surface and to be easily
recovered.
4. The thin sections could be first observed by optic microscopy
in order to guarantee preservation of the tissue structure and
quality.
5. A chloroform drop should be included in the water for section
stretching before picking.
6. Fish sections with an instrument containing the selected grid.
Tissue to be observed should be localized in the center of the
grid. Sections should be put in nickel grids (and not copper).
7. Leave the sections to dry for 24 h before staining.
3.3 Grid Staining
1. Place a piece of wax-paper into a petri dish.
2. Invert grids section side down onto drops of uranyl acetate
solution for 15 min in the dark (see Notes 5 and 6).
3. Wash gently in a stream of distilled water and blot dry.
4. Invert grids section side down onto drops of Reynolds lead
stain (5 min) in the presence of NaOH pellets (in order to
absorb CO 2 ). A petri dish with cover is a suitable place to avoid
gas exchange. Lead stain produces a precipitate on exposure to
carbon dioxide. Be careful and avoid breathing into samples
during staining (see Note 7).
5. Wash gently in a stream of distilled water and blot dry (see
Notes 8 and 9).
6. Store the staining grids in appropriate racks, properly identified, until microscopic observation (see Note 9).
3.4 TEM
Observations
1. To initiate TEM observation of the different grids, the microscope should be property adjusted following the Microscopy
Unit instructions and working protocols that may vary for each
particular microscope.
2. As a general procedure, the grid should be mounted over the
sample holder, properly adjusted to the microscope allowing
then perform the vacuum.
3. Selection of appropriate voltage and brightness, and searching
for observation cell sections of interest usually at low magnifications to get a wider image of the sections. Increase
Structure of Plastid-Containing Carotenoids from Fruits
241