3 Methods
3.1 Infiltration
1. Harvest fully expanded leaves from grapevine and coffee shoots
to obtain approximately 25 g and 10 g of fresh weight material,
respectively. Remove the middle and lateral veins using a scalpel
(see Fig. 1a, b, Note 7).
2. Cut leaves into pieces of around 2 cm
2 to a glass or plastic
beaker (see Fig. 1, Note 8) kept on ice. Transfer the leaf pieces
to a large glass Kitasato flask filled with ice-cold infiltration
buffer (see Fig. 2a, Note 9).
3. Apply vacuum to the Kitasato flask employing a vacuum pump.
A final pressure of about 25 kPa around 187.52 mmHg is
sufficient for infiltration. A total infiltration time of 3 min (six
periods of 30 s) was applied.
4. Slowly release the vacuum by releasing the vacuum tube on the
Kitasato flask. During vacuum release, the leaves are slowly
infiltrated with the infiltration buffer. During this process, leaf
tissue becomes water-soaked and dark in color (see Fig. 2b).
Fig. 1 Preparation of grapevine (a) and coffee (b) leaves for infiltration. Leaves are cut with a scalpel in 2 cm
2
squares after removing the middle and lateral veins (grapevine, c) and the middle vein (coffee, d)
52
Andreia Figueiredo and Leonor Guerra-Guimara ˜ es
3.1 Infiltration
1. Harvest fully expanded leaves from grapevine and coffee shoots
to obtain approximately 25 g and 10 g of fresh weight material,
respectively. Remove the middle and lateral veins using a scalpel
(see Fig. 1a, b, Note 7).
2. Cut leaves into pieces of around 2 cm
2 to a glass or plastic
beaker (see Fig. 1, Note 8) kept on ice. Transfer the leaf pieces
to a large glass Kitasato flask filled with ice-cold infiltration
buffer (see Fig. 2a, Note 9).
3. Apply vacuum to the Kitasato flask employing a vacuum pump.
A final pressure of about 25 kPa around 187.52 mmHg is
sufficient for infiltration. A total infiltration time of 3 min (six
periods of 30 s) was applied.
4. Slowly release the vacuum by releasing the vacuum tube on the
Kitasato flask. During vacuum release, the leaves are slowly
infiltrated with the infiltration buffer. During this process, leaf
tissue becomes water-soaked and dark in color (see Fig. 2b).
Fig. 1 Preparation of grapevine (a) and coffee (b) leaves for infiltration. Leaves are cut with a scalpel in 2 cm
2
squares after removing the middle and lateral veins (grapevine, c) and the middle vein (coffee, d)
52
Andreia Figueiredo and Leonor Guerra-Guimara ˜ es