After freezing, the Sapphire disc was removed from the middle plate in anhydrous acetone at -90°C and immediately transferred into a 2 ml Eppendorf tube containing 2% OsO4 in anhydrous acetone, precooled to -90 °C in an Leica EM AFS2 freeze-substitution unit. Samples were substituted for 8 h at -90°C, 8 h at -60°C, 8 h at -30°C, and 1 h at 0°C with periodic temperature transition gradients of 30°C/h.
Samples were then washed twice with anhydrous acetone at 4°C, immersed in 33% Epon/Araldite in anhydrous acetone at 4°C overnight, followed by 66% Epon/Araldite in anhydrous acetone at 4°C for 6 h, and finally in 100% Epon/Araldite at room temperature for 2 h prior to polymerization at 60°C for at least 24 h in a 1.5 ml Eppendorf tube. Sections were post-stained with uranyl acetate and lead citrate.
NOTE: The use of ethanol as a synchronization fluid during high-pressure freezing is not necessary for cell culture monolayers on Sapphire discs.
A simplified sandwich configuration can be used to provide similar results by placing the Sapphire disc in a CLEM middle plate with cells facing up covered with an aluminium specimen carrier dipped in 1-hexadecene with the 100 μm cavity facing the cells.