3. PBS-T-0.1%: Dilute 1 mL of 10Â PBS with 9 mL of water and
add 10 μL of Tween-20.
4. PBS-T-0.5%: Dilute 100 mL of 10Â PBS with 900 mL of
distilled water and add 0.5 mL of Tween-20.
5. Blocking buffer: 5% (w/v) non-fat milk in PBS-T-0.1%.
6. Primary antibody in blocking buffer.
7. Secondary antibody conjugated to gold particles (5, 10, or
15 nm in diameter) diluted (1:10) in blocking buffer.
3 Methods
3.1 Plant Material
3.1.1 Arabidopsis
Seedlings: Root Tips and
Cotyledons
The area close to the root tip contains dividing, elongating, and
differentiating cells and it is usually chosen for electron microscopy
studies (Fig. 1a). To obtain root tips from 7 to 10-day-old seedlings, it is best to germinate seeds on 0.8% agar plates containing ½
strength MS basal medium. Cotyledons can also be successfully
used for TEM studies (Fig. 1b–d) but it is recommendable to
grow seedling in liquid medium for this purpose (see Note 4).
1. Place Arabidopsis seeds in plastic tube and add 10% bleach for
5 min, mixing occasionally.
2. Remove bleach using sterile glass Pasteur pipette or sterile
1 mL pipette tips and rinse three times with sterile water.
3. Add 70% ethanol, mix, and discard after 5 min.
4. Rinse three times with sterile water and place seeds on either
0.8% agar plates supplemented with ½ strength MS for processing of root tips or in small Petri dishes containing 5 mL of
liquid ½ strength MS for processing cotyledons.
3.1.2 Developing Seeds
Developing seeds have also been proven to be excellent material for
high-pressure freezing [12–14]. They can be used to study structural cellular details of both developing embryo and endosperm as
well as the seed coat layers. These are ideal tissues to study cellularization of the nuclear endosperm (approximately 5–6 days after
pollination) and cytokinesis and accumulation of storage compounds in embryo cells.
3.1.3 Developing Anthers
The process of meiosis, microspore and pollen formation, pollen
wall differentiation, and tapetum maturation has been studied in
wild-type and mutant Arabidopsis plants using high-pressure frozen samples [15–19] (Fig. 2). In an Arabidopsis plant, the spiral
arrangement of floral buds from older to younger allows researchers to identify all stages of anther development in a single
inflorescence.
340
Marisa S. Otegui
add 10 μL of Tween-20.
4. PBS-T-0.5%: Dilute 100 mL of 10Â PBS with 900 mL of
distilled water and add 0.5 mL of Tween-20.
5. Blocking buffer: 5% (w/v) non-fat milk in PBS-T-0.1%.
6. Primary antibody in blocking buffer.
7. Secondary antibody conjugated to gold particles (5, 10, or
15 nm in diameter) diluted (1:10) in blocking buffer.
3 Methods
3.1 Plant Material
3.1.1 Arabidopsis
Seedlings: Root Tips and
Cotyledons
The area close to the root tip contains dividing, elongating, and
differentiating cells and it is usually chosen for electron microscopy
studies (Fig. 1a). To obtain root tips from 7 to 10-day-old seedlings, it is best to germinate seeds on 0.8% agar plates containing ½
strength MS basal medium. Cotyledons can also be successfully
used for TEM studies (Fig. 1b–d) but it is recommendable to
grow seedling in liquid medium for this purpose (see Note 4).
1. Place Arabidopsis seeds in plastic tube and add 10% bleach for
5 min, mixing occasionally.
2. Remove bleach using sterile glass Pasteur pipette or sterile
1 mL pipette tips and rinse three times with sterile water.
3. Add 70% ethanol, mix, and discard after 5 min.
4. Rinse three times with sterile water and place seeds on either
0.8% agar plates supplemented with ½ strength MS for processing of root tips or in small Petri dishes containing 5 mL of
liquid ½ strength MS for processing cotyledons.
3.1.2 Developing Seeds
Developing seeds have also been proven to be excellent material for
high-pressure freezing [12–14]. They can be used to study structural cellular details of both developing embryo and endosperm as
well as the seed coat layers. These are ideal tissues to study cellularization of the nuclear endosperm (approximately 5–6 days after
pollination) and cytokinesis and accumulation of storage compounds in embryo cells.
3.1.3 Developing Anthers
The process of meiosis, microspore and pollen formation, pollen
wall differentiation, and tapetum maturation has been studied in
wild-type and mutant Arabidopsis plants using high-pressure frozen samples [15–19] (Fig. 2). In an Arabidopsis plant, the spiral
arrangement of floral buds from older to younger allows researchers to identify all stages of anther development in a single
inflorescence.
340
Marisa S. Otegui