Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue
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Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue. / Studer, Daniel; Zhao, Shanting; Chai, Xuejun; Jonas, Peter; Graber, Werner; Nestel, Sigrun; Frotscher, Michael.
in: NAT PROTOC, Jahrgang 9, Nr. 6, 01.01.2014, S. 1480-95.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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T1 - Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue
AU - Studer, Daniel
AU - Zhao, Shanting
AU - Chai, Xuejun
AU - Jonas, Peter
AU - Graber, Werner
AU - Nestel, Sigrun
AU - Frotscher, Michael
PY - 2014/1/1
Y1 - 2014/1/1
N2 - Electron microscopy (EM) allows for the simultaneous visualization of all tissue components at high resolution. However, the extent to which conventional aldehyde fixation and ethanol dehydration of the tissue alter the fine structure of cells and organelles, thereby preventing detection of subtle structural changes induced by an experiment, has remained an issue. Attempts have been made to rapidly freeze tissue to preserve native ultrastructure. Shock-freezing of living tissue under high pressure (high-pressure freezing, HPF) followed by cryosubstitution of the tissue water avoids aldehyde fixation and dehydration in ethanol; the tissue water is immobilized in ∼50 ms, and a close-to-native fine structure of cells, organelles and molecules is preserved. Here we describe a protocol for HPF that is useful to monitor ultrastructural changes associated with functional changes at synapses in the brain but can be applied to many other tissues as well. The procedure requires a high-pressure freezer and takes a minimum of 7 d but can be paused at several points.
AB - Electron microscopy (EM) allows for the simultaneous visualization of all tissue components at high resolution. However, the extent to which conventional aldehyde fixation and ethanol dehydration of the tissue alter the fine structure of cells and organelles, thereby preventing detection of subtle structural changes induced by an experiment, has remained an issue. Attempts have been made to rapidly freeze tissue to preserve native ultrastructure. Shock-freezing of living tissue under high pressure (high-pressure freezing, HPF) followed by cryosubstitution of the tissue water avoids aldehyde fixation and dehydration in ethanol; the tissue water is immobilized in ∼50 ms, and a close-to-native fine structure of cells, organelles and molecules is preserved. Here we describe a protocol for HPF that is useful to monitor ultrastructural changes associated with functional changes at synapses in the brain but can be applied to many other tissues as well. The procedure requires a high-pressure freezer and takes a minimum of 7 d but can be paused at several points.
KW - Brain
KW - Cryoelectron Microscopy
KW - Neurons
KW - Pressure
KW - Synapses
U2 - 10.1038/nprot.2014.099
DO - 10.1038/nprot.2014.099
M3 - SCORING: Journal article
C2 - 24874814
VL - 9
SP - 1480
EP - 1495
JO - NAT PROTOC
JF - NAT PROTOC
SN - 1754-2189
IS - 6
ER -