The grids were rendered hydrophilic with a Fischione plasma cleaner for 20 s in a specially adapted stage, then immediately used for sample preparation in a vitrobot (FEI, Netherlands) at 25°C and 100% humidity, blotted for 2 3 s, and rapidly plunged into liquid ethane maintained at −180°C.
Frozen-hydrated specimens were prepared on plasma-cleaned Quantifoil R1.2/1.3 holey carbon EM grids (Quantifoil, Großlöbichau, Germany) using a Vitrobot Mark III (FEI, Hillsboro, OR, USA) 5 s blotting time, 85% humidity and −5 mm blotting offset.
The solutions were initially loaded on a copper grid with Lacey carbon film (Electron Microscopy Sciences, Hatfield, PA) in the controlled humidity chamber and were blotted by the filter papers that were mounted on the Vitrobot from both sides of the grid.
The specimen was then blotted under 95% humidity, immediately transferred into liquid ethane, and stored in liquid nitrogen environment.
Grids were blotted in 100% humidity at 4°C for 5 s and plunge-frozen into liquid ethane cooled by liquid nitrogen using a Vitrobot (FEI, Hillsboro, OR).
Grids were blotted in 100% humidity at 4°C for 5 s before being plunge-frozen into liquid ethane bathed in liquid nitrogen inside a Vitrobot (FEI, Hillsboro, OR).
The slide was placed into a petri dish with moistened blotting paper to maintain humidity, and incubated for 12 hrs (37°C).
The grid was blotted in an FEI Vitrobot Mark IV (FEI Company, Hilsboro, OR) using 4 s blotting time and blotting force 2 with 100% humidity at 283 K, and then plunged into the chilled liquid ethane.
The |grids were blotted in an FEI Vitrobot using a 2.5 s blotting time at 70% humidity and 10°C and plunge-frozen in liquid ethane.
