9. There are commercial and custom-made devices to stain multiple grids at once. For example, the Grid Staining Matrix System
from Ted Pella can handle up to 25 grids ensuring reduced
handling of grids and equal staining and rinsing times.
Acknowledgement
This work was supported by NSF MCB1614965 to M.S.O.
References
1. Gilkey JC, Staehelin LA (1986) Advances in
ultrarapid freezing for the preservation of cellular ultrastructure. J Electron Microsc Tech
3:177–210
2. Watanabe S et al (2013) Ultrafast endocytosis
at mouse hippocampal synapses. Nature
504:242–247
3. Watanabe S (2016) Flash-and-freeze: coordinating optogenetic stimulation with rapid
freezing to visualize membrane dynamics at
synapses with millisecond resolution. Front
Synaptic Neurosci 8:24
4. Studer D, Michel M, Mu ¨ller M (1989) Highpressure freezing comes of age. Scan Microsc
Suppl 3:253–269
5. Kiss JZ, Staehelin LA (1995) High pressure
freezing. In: Severs NJ, Shotton DM (eds)
Rapid freezing, freeze fracture and deep etching. Wiley-Liss, New York, pp 89–104
6. Hess MW (2003) Of plants and other pets:
practical aspectes of freeze-substitution and
resin embedding. J Microsc 212:44–52
7. McDonald K (1999) High-pressure freezing for
preservation of high resolution fine structure and
antigenicity for immunolabeling. In: Hajibagheri N (ed) Electron microscopy methods and
protocols (methods in molecular biology, vol.
117. Humana Press, Totowa, pp 77–97
8. Otegui MS (2011) Electron tomography and
immunogold labelling as tools to analyse de
novo assembly of plant cell walls. Methods
Mol Biol 715:123–140
9. Otegui MS (2014) Electron tomography of
plant cells. In: Assmann S, Liu B (eds) Cell
biology. Springer, New York, pp 1–14
10. Otegui MS (2014) Plant endosomes—methods and protocols. Humana Press, New York
11. Woog I et al (2012) Correlative light and electron microscopy of intermediate stages of meiotic spindle assembly in the early
Caenorhabditis elegans embryo. Methods Cell
Biol 111:223–234
12. Otegui MS et al (2001) Three-dimensional
analysis of syncytial-type cell plates during
endosperm cellularization visualized by high
resolution electron tomography. Plant Cell
13:2033–2051
13. Otegui MS, Staehelin LA (2000) Cytokinesis
in flowering plants: more than one way to
divide a cell. Curr Opin Plant Biol 3:493–502
14. Otegui MS, Staehelin LA (2000) Syncytialtype cell plates: a novel kind of cell plate
involved in endosperm cellularization of Arabidopsis. Plant Cell 12:933–947
15. Quilichini TD, Douglas CJ, Samuels AL
(2014) New views of tapetum ultrastructure
and pollen exine development in Arabidopsis
thaliana. Ann Bot 114:1189–1201
16. Quilichini TD et al (2010) ATP-binding cassette transporter G26 is required for male fertility and pollen exine formation in
Arabidopsis. Plant Physiol 154:678–690
17. Quilichini TD, Samuels AL, Douglas CJ
(2014) ABCG26-mediated polyketide trafficking and hydroxycinnamoyl spermidines contribute to pollen wall exine formation in
Arabidopsis. Plant Cell 26:4483–4498
18. Otegui MS, Staehelin LA (2004) Electron
tomographic analysis of post-meiotic cytokinesis during pollen development in Arabidopsis
thaliana. Planta 218:501–515
19. Backues SK et al (2010) The Arabidopsis
dynamin-related protein2 family is essential
for gametophyte development. Plant Cell
22:3218–3231
20. Giddings TH (2003) Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples. J
Microsc 212:53–61
21. McDonald KL (2014) Rapid embedding
methods into epoxy and LR white resins for
morphological and immunological analysis of
cryofixed biological specimens. Microsc Microanal 20:152–163
22. Reipert S et al (2018) Agitation modules:
flexible means to accelerate automated freeze
substitution.
J
Histochem
Cytochem
66:903–921
High-Pressure Freezing and Freeze Substitution for Transmission Electron. . .
347
from Ted Pella can handle up to 25 grids ensuring reduced
handling of grids and equal staining and rinsing times.
Acknowledgement
This work was supported by NSF MCB1614965 to M.S.O.
References
1. Gilkey JC, Staehelin LA (1986) Advances in
ultrarapid freezing for the preservation of cellular ultrastructure. J Electron Microsc Tech
3:177–210
2. Watanabe S et al (2013) Ultrafast endocytosis
at mouse hippocampal synapses. Nature
504:242–247
3. Watanabe S (2016) Flash-and-freeze: coordinating optogenetic stimulation with rapid
freezing to visualize membrane dynamics at
synapses with millisecond resolution. Front
Synaptic Neurosci 8:24
4. Studer D, Michel M, Mu ¨ller M (1989) Highpressure freezing comes of age. Scan Microsc
Suppl 3:253–269
5. Kiss JZ, Staehelin LA (1995) High pressure
freezing. In: Severs NJ, Shotton DM (eds)
Rapid freezing, freeze fracture and deep etching. Wiley-Liss, New York, pp 89–104
6. Hess MW (2003) Of plants and other pets:
practical aspectes of freeze-substitution and
resin embedding. J Microsc 212:44–52
7. McDonald K (1999) High-pressure freezing for
preservation of high resolution fine structure and
antigenicity for immunolabeling. In: Hajibagheri N (ed) Electron microscopy methods and
protocols (methods in molecular biology, vol.
117. Humana Press, Totowa, pp 77–97
8. Otegui MS (2011) Electron tomography and
immunogold labelling as tools to analyse de
novo assembly of plant cell walls. Methods
Mol Biol 715:123–140
9. Otegui MS (2014) Electron tomography of
plant cells. In: Assmann S, Liu B (eds) Cell
biology. Springer, New York, pp 1–14
10. Otegui MS (2014) Plant endosomes—methods and protocols. Humana Press, New York
11. Woog I et al (2012) Correlative light and electron microscopy of intermediate stages of meiotic spindle assembly in the early
Caenorhabditis elegans embryo. Methods Cell
Biol 111:223–234
12. Otegui MS et al (2001) Three-dimensional
analysis of syncytial-type cell plates during
endosperm cellularization visualized by high
resolution electron tomography. Plant Cell
13:2033–2051
13. Otegui MS, Staehelin LA (2000) Cytokinesis
in flowering plants: more than one way to
divide a cell. Curr Opin Plant Biol 3:493–502
14. Otegui MS, Staehelin LA (2000) Syncytialtype cell plates: a novel kind of cell plate
involved in endosperm cellularization of Arabidopsis. Plant Cell 12:933–947
15. Quilichini TD, Douglas CJ, Samuels AL
(2014) New views of tapetum ultrastructure
and pollen exine development in Arabidopsis
thaliana. Ann Bot 114:1189–1201
16. Quilichini TD et al (2010) ATP-binding cassette transporter G26 is required for male fertility and pollen exine formation in
Arabidopsis. Plant Physiol 154:678–690
17. Quilichini TD, Samuels AL, Douglas CJ
(2014) ABCG26-mediated polyketide trafficking and hydroxycinnamoyl spermidines contribute to pollen wall exine formation in
Arabidopsis. Plant Cell 26:4483–4498
18. Otegui MS, Staehelin LA (2004) Electron
tomographic analysis of post-meiotic cytokinesis during pollen development in Arabidopsis
thaliana. Planta 218:501–515
19. Backues SK et al (2010) The Arabidopsis
dynamin-related protein2 family is essential
for gametophyte development. Plant Cell
22:3218–3231
20. Giddings TH (2003) Freeze-substitution protocols for improved visualization of membranes in high-pressure frozen samples. J
Microsc 212:53–61
21. McDonald KL (2014) Rapid embedding
methods into epoxy and LR white resins for
morphological and immunological analysis of
cryofixed biological specimens. Microsc Microanal 20:152–163
22. Reipert S et al (2018) Agitation modules:
flexible means to accelerate automated freeze
substitution.
J
Histochem
Cytochem
66:903–921
High-Pressure Freezing and Freeze Substitution for Transmission Electron. . .
347