To begin preparing the electron microscopy grids for cellular cryotomography, place the grid, loaded with perforated carbon support film in a glow discharge device with the carbon side of the grid facing up. Then, glow discharge the grid at 15 milliamperes, under 0.39 millibar atmospheric pressure for 45 seconds. Once done, place the grid in a clean container, lined with filter paper.
Next, transfer the grid along with fibronectin, phosphate buffered saline or PBS, plates and tweezers to a biosafety cabinet. Using a micro pipette, cast two generously sized dots of bovine fibronectin onto a sterile surface like a six well plate lid. Ensure the dots are large enough to envelop the entire grid.
Then, obtain five by 15 tweezers and treat both sides of the grid with the fibronectin adherence solution. Once treated with bovine fibronectin, the grid will become sticky. Gently touch the non-carbon surface of the grid to the bottom of an empty well in a six well plate and release the tweezers to attach the grid to the new surface.
Next, incubate the grid for 30 minutes in the fibronectin adherence solution. To prevent the grids from drying, add one to two drops of adherence solution on top of the grids, using a micro pipette every 15 minutes. Following the incubation, remove the excess adherence solution with a micro pipette.
Wash the grids by adding drops of PBS directly on top of the grids, using the micro pipette two to three times. Sterilize the grids under UV light in a biosafety cabinet for one hour. Position the grids as close to the UV as possible for maximum sterilization.
Prevent the grids from drying during sterilization by micro pipetting one to two drops of PBS on top of the grid every 10 minutes.
