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.
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We thank K. Miller for her help with Supplementary Video 1 and N. Brose (Max Planck Institute for Experimental Medicine) for providing the Munc13-1 mutants. This study was supported by grants from the Deutsche Forschungsgemeinschaft (SFB 780 and FR 620/12-1 to M.F.) and the Swiss National Foundation (grant no. 3100AO_118394 to D.S.). M.F. is Senior Research Professor for Neuroscience of the Hertie Foundation.
D.S. was involved in the development of EM PACT and receives royalties from Leica Microsystems (Vienna, Austria).
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Studer, D., Zhao, S., Chai, X. et al. Capture of activity-induced ultrastructural changes at synapses by high-pressure freezing of brain tissue. Nat Protoc 9, 1480–1495 (2014). https://doi.org/10.1038/nprot.2014.099