2.6 Cryo-Electron
Tomography
1. Microscope: 300 kV cryo-TEM.
2. Data Collection Software: Batch tomography collection software, such as Tomography (Thermo Fisher Scientific), SerialEM (Boulder, CO), Leginon (NRAMM), or UCSFTomo
(UCSF, CA).
2.7 Image
Processing Software
1. Reconstruction: IMOD 4.9.12 (UC Boulder, CO).
2. Segmentation and Visualization: EMAN 2.31 (Baylor College
of Medicine, TX) and Amira (Thermo Fisher Scientific).
3 Methods
3.1 Grid Preparation
1. UV and ethanol sterilize a biosafety cabinet.
2. Ethanol sterilize a glass slide and a 100 mm petri dish. Place the
slide inside the petri dish (see Note 4).
3. In the biosafety cabinet, use autoclaved forceps to remove the
grids from the storage box and set them carbon-side up on the
glass slide.
4. Examine grids’ carbon supports under a stereo microscope to
ensure that the majority of the surface is intact.
5. Glow discharge grids to produce a hydrophilic surface (see
Note 5).
6. Carefully place the glass top on the petri dish before transfering
the grids into the biosafety cabinet. UV sterilize forceps and
grids for 5–10 min.
3.2 Poly-D-Lysine
Coating
1. Dilute Poly-D-lysine (PDL) to 100 μg/mL in Milli-Q H 2 O. Filter sterilize PDL in biosafety cabinet through a 0.2 μm PES
vacuum filter.
2. Add enough PDL solution to completely cover the bottom of
the dish. 2 mL is sufficient in a 35 mm glass bottom dish.
3. Using forceps, submerge the grids carbon-side up in the PDL
solution and place on the glass bottom of the dish (see Note 6).
4. Place dish containing grids into a 5% CO
2
, 37
C incubator
overnight.
5. The next morning, wash grids three times with 2 mL of
autoclave-sterilized Milli-Q H 2 O, being careful not to disturb
the grids. The wash step is very important; excess PDL in the
growth media can inhibit neurite outgrowth.
6. Leave grids in water or growth media and place back in the
incubator until neurons are to be plated.
28
Ryan K. Hylton et al.
Tomography
1. Microscope: 300 kV cryo-TEM.
2. Data Collection Software: Batch tomography collection software, such as Tomography (Thermo Fisher Scientific), SerialEM (Boulder, CO), Leginon (NRAMM), or UCSFTomo
(UCSF, CA).
2.7 Image
Processing Software
1. Reconstruction: IMOD 4.9.12 (UC Boulder, CO).
2. Segmentation and Visualization: EMAN 2.31 (Baylor College
of Medicine, TX) and Amira (Thermo Fisher Scientific).
3 Methods
3.1 Grid Preparation
1. UV and ethanol sterilize a biosafety cabinet.
2. Ethanol sterilize a glass slide and a 100 mm petri dish. Place the
slide inside the petri dish (see Note 4).
3. In the biosafety cabinet, use autoclaved forceps to remove the
grids from the storage box and set them carbon-side up on the
glass slide.
4. Examine grids’ carbon supports under a stereo microscope to
ensure that the majority of the surface is intact.
5. Glow discharge grids to produce a hydrophilic surface (see
Note 5).
6. Carefully place the glass top on the petri dish before transfering
the grids into the biosafety cabinet. UV sterilize forceps and
grids for 5–10 min.
3.2 Poly-D-Lysine
Coating
1. Dilute Poly-D-lysine (PDL) to 100 μg/mL in Milli-Q H 2 O. Filter sterilize PDL in biosafety cabinet through a 0.2 μm PES
vacuum filter.
2. Add enough PDL solution to completely cover the bottom of
the dish. 2 mL is sufficient in a 35 mm glass bottom dish.
3. Using forceps, submerge the grids carbon-side up in the PDL
solution and place on the glass bottom of the dish (see Note 6).
4. Place dish containing grids into a 5% CO
2
, 37
C incubator
overnight.
5. The next morning, wash grids three times with 2 mL of
autoclave-sterilized Milli-Q H 2 O, being careful not to disturb
the grids. The wash step is very important; excess PDL in the
growth media can inhibit neurite outgrowth.
6. Leave grids in water or growth media and place back in the
incubator until neurons are to be plated.
28
Ryan K. Hylton et al.