P. tricornutum cells expressing different types of GFP fusion proteins were harvested via centrifugation at 1,500xg and cryoimmobilized by high-pressure freezing on gold carriers (Leica EM PACT2, Leica Microsystems GmbH, Vienna, Austria). Subsequently, the cells were freeze-substituted with acetone in combination with 2% OsO4, 0.25% uranyl acetate and 5% H2O. Freeze substitution was carried out in the automated Leica EM AFS2 unit (Leica Microsystems GmbH, Vienna, Austria) at -90°C for 4 h, -60°C for 8 h, -30°C for 8 h and then held at 0°C for at least 3 h ...
For more details please see:
Dr. Kathrin Bolte and Dr. Andreas Klingl
Zellbiologie & Elektronenmikroskopie
AG Prof. Uwe-G. Maier
Philipps Universität Marburg, Marburg
The website of the EM facility is:
<media 50974 - external-link-new-window "Opens external link in new window">Download the Application Note</media>