3. In the options section of the screen, select the following: “Use
foot pedal”, “Humidifier off during process”, and “Skip grid
transfers.”
4. Cool the copper insert, and Styrofoam cup with liquid nitrogen. Once cool, fill the inner chamber to the top with liquid
ethane by putting a 1000 μL pipette tip in the ethane hose,
pressing the tip into the corner of the ethane cup, and slowly
filling with ethane until there is a convex liquid surface at the
rim of the cup. Quickly remove the pipette tip and allow time
for the ethane to cool properly (see Note 9).
5. Label cryo grid boxes and submerge in liquid nitrogen. On the
aluminum platform in the Styrofoam cup, there are cutouts
where the grid boxes will sit in place.
6. Transport culture dishes containing grids to the plunge freezer
and place on a 37
C heating block (or bench top incubator, if
available).
7. Dilute BSA-coated gold fiducials 1:4 in culture media from
dish and maintain at 37
C on the heating block.
8. Carefully pick up grid from the bottom of dish with tweezers,
keeping the cell-side up. Use clip on tweezers to lock them in
the clamped position.
9. Set tweezers down carefully (cell-side up), and pipette 3 μL of
fiducial gold/media mixture on to the grid from the edge,
being careful not to touch and bend the grid with the
pipette tip.
10. Attach the tweezers to the plunging apparatus with the cells
facing away from the side that will be blotted. For example: if
blotting from the right, cells should be facing the left side of
the blotting chamber.
Fig. 3 Single-sided manual blotting in the Vitrobot. (a) Cut a small rectangle of filter paper and bend toward the
center, lengthwise. (b) Use a set of long sturdy forceps to reach through the open door on the side of the
Vitrobot chamber and make sure the paper is aligned with the back-side of the grid. (c) Press the blotting
paper flush against the back of the grid and watch the water wick away. Counting 2 s is a good place to start
with neurons (or other adherent cells)
Cryotomography of Neurons
31
foot pedal”, “Humidifier off during process”, and “Skip grid
transfers.”
4. Cool the copper insert, and Styrofoam cup with liquid nitrogen. Once cool, fill the inner chamber to the top with liquid
ethane by putting a 1000 μL pipette tip in the ethane hose,
pressing the tip into the corner of the ethane cup, and slowly
filling with ethane until there is a convex liquid surface at the
rim of the cup. Quickly remove the pipette tip and allow time
for the ethane to cool properly (see Note 9).
5. Label cryo grid boxes and submerge in liquid nitrogen. On the
aluminum platform in the Styrofoam cup, there are cutouts
where the grid boxes will sit in place.
6. Transport culture dishes containing grids to the plunge freezer
and place on a 37
C heating block (or bench top incubator, if
available).
7. Dilute BSA-coated gold fiducials 1:4 in culture media from
dish and maintain at 37
C on the heating block.
8. Carefully pick up grid from the bottom of dish with tweezers,
keeping the cell-side up. Use clip on tweezers to lock them in
the clamped position.
9. Set tweezers down carefully (cell-side up), and pipette 3 μL of
fiducial gold/media mixture on to the grid from the edge,
being careful not to touch and bend the grid with the
pipette tip.
10. Attach the tweezers to the plunging apparatus with the cells
facing away from the side that will be blotted. For example: if
blotting from the right, cells should be facing the left side of
the blotting chamber.
Fig. 3 Single-sided manual blotting in the Vitrobot. (a) Cut a small rectangle of filter paper and bend toward the
center, lengthwise. (b) Use a set of long sturdy forceps to reach through the open door on the side of the
Vitrobot chamber and make sure the paper is aligned with the back-side of the grid. (c) Press the blotting
paper flush against the back of the grid and watch the water wick away. Counting 2 s is a good place to start
with neurons (or other adherent cells)
Cryotomography of Neurons
31