it also provides the means to capture elusive intermediates in highly
dynamic cellular processes [2, 3]. Cryofixation at atmospheric pressure is limited to samples less than ~15 μm thick due to damage
from ice crystal formation. High-pressure freezing produces
vitreous ice in samples up to ~200 μm thick [4, 5]. For most
applications, the high-pressure frozen sample is then subjected to
freeze-substitution, in which the water is gradually replaced with an
organic solvent (e.g., acetone) with fixatives at low temperatures
(e.g., À90
C) and then embedded in resin for sectioning [6]. This
method is applicable to a wide variety of cells, tissues, and organisms [7–11] and can be used for structural work and for
immunogold-based detection of proteins and other molecules.
The superb cellular preservation achieved by high-pressure
freezing is frequently coupled to other powerful approaches in
cell biology, such as correlative light and electron microscopy
(CLEM) and three-dimensional reconstruction by electron tomography. For CLEM, fluorescent signals in the biological samples can
be imaged before or after high-pressure freezing in such a way that
the same fluorescent signal can be precisely mapped onto the TEM
image.
This chapter includes protocols for TEM imaging and
immunogold-labeling of high-pressure frozen-freeze substituted
Arabidopsis material, including root tips, cotyledons, developing
seeds, and anthers.
2 Materials
2.1 Plant-Related
Materials
1. Sterile hood.
2. 10% Bleach.
3. 70% Ethanol.
4. Sterile water.
5. Sterile glass Pasteur pipettes or sterile 1 mL pipette tips.
6. Arabidopsis seeds.
7. 0.8% Agar plates containing ½ strength: 8 g of agar plus 2.2 g
of Murashige and Skoog (MS) basal medium powder per liter
of solution.
8. Plastic Petri dishes (5 cm in diameter) containing 5 mL of ½
strength MS liquid medium.
9. Arabidopsis plants in reproductive stage with floral buds and
siliques containing developing seeds (between 5 and 10 days
after pollination).
2.2 High-Pressure
Freezing
1. High-pressure freezer (Leica EM ICE, Bal-tec/ABRA Fluid
AG HPM 010, or M.Wohlwend HPF Compact 02).
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Marisa S. Otegui
dynamic cellular processes [2, 3]. Cryofixation at atmospheric pressure is limited to samples less than ~15 μm thick due to damage
from ice crystal formation. High-pressure freezing produces
vitreous ice in samples up to ~200 μm thick [4, 5]. For most
applications, the high-pressure frozen sample is then subjected to
freeze-substitution, in which the water is gradually replaced with an
organic solvent (e.g., acetone) with fixatives at low temperatures
(e.g., À90
C) and then embedded in resin for sectioning [6]. This
method is applicable to a wide variety of cells, tissues, and organisms [7–11] and can be used for structural work and for
immunogold-based detection of proteins and other molecules.
The superb cellular preservation achieved by high-pressure
freezing is frequently coupled to other powerful approaches in
cell biology, such as correlative light and electron microscopy
(CLEM) and three-dimensional reconstruction by electron tomography. For CLEM, fluorescent signals in the biological samples can
be imaged before or after high-pressure freezing in such a way that
the same fluorescent signal can be precisely mapped onto the TEM
image.
This chapter includes protocols for TEM imaging and
immunogold-labeling of high-pressure frozen-freeze substituted
Arabidopsis material, including root tips, cotyledons, developing
seeds, and anthers.
2 Materials
2.1 Plant-Related
Materials
1. Sterile hood.
2. 10% Bleach.
3. 70% Ethanol.
4. Sterile water.
5. Sterile glass Pasteur pipettes or sterile 1 mL pipette tips.
6. Arabidopsis seeds.
7. 0.8% Agar plates containing ½ strength: 8 g of agar plus 2.2 g
of Murashige and Skoog (MS) basal medium powder per liter
of solution.
8. Plastic Petri dishes (5 cm in diameter) containing 5 mL of ½
strength MS liquid medium.
9. Arabidopsis plants in reproductive stage with floral buds and
siliques containing developing seeds (between 5 and 10 days
after pollination).
2.2 High-Pressure
Freezing
1. High-pressure freezer (Leica EM ICE, Bal-tec/ABRA Fluid
AG HPM 010, or M.Wohlwend HPF Compact 02).
338
Marisa S. Otegui