process the data. Though the methods described are not exhaustive
in terms of all the possible options and microscope configurations,
much of this methodology can serve as a resource for others venturing into the study of 3D cellular architecture in general.
2 Materials
2.1 Grid Preparation
1. Sterilized, fine-tipped tweezers (Dumont No. 5).
2. 200 mesh gold QUANTIFOIL R 2/2 holey carbon grids. See
Note 1.
3. Glow Discharge System.
4. Glass slide and glass petri dish. Manufacturer and size of the
slide and dish do not matter, as long as the dish is large enough
to hold the slide.
5. 70% ethanol.
2.2 PDL Coating
1. Poly-D-Lysine solution.
2. 0.2 μm PES vacuum filter.
2.3 Neuronal Culture
1. Hippocampal Neurons. We use a Complete Kit for E18 Sprague Dawley rat hippocampus (BrainBits). See Note 2.
2. 35 mm glass bottom cell culture dishes. See Note 3.
3. Culture Media: We use NbActiv4 media (BrainBits) with 1%
Pen-Strep, but standard neurobasal media and other variants
have been used successfully (see Note 2).
4. Trypan Blue Solution, 0.4%.
2.4 Fiducial Gold
1. Lyophilized BSA.
2. 10 nm colloidal gold nanoparticles.
2.5 Vitrification
1. Whatman Grade 1 Qualitative Filter Paper; 55 mm circles.
2. Plunge Freezer. We use the Thermo Fisher Scientific Vitrobot
Mark IV, but any should be able to work, even completely
manual setups without strict environmental controls.
3. Ethane Canister.
4. Liquid Nitrogen.
5. Liquid Nitrogen dewars for transport and storage.
6. CryoEM grid storage boxes.
7. Long (>6 in.) pair of blunt tweezers.
Cryotomography of Neurons
27
in terms of all the possible options and microscope configurations,
much of this methodology can serve as a resource for others venturing into the study of 3D cellular architecture in general.
2 Materials
2.1 Grid Preparation
1. Sterilized, fine-tipped tweezers (Dumont No. 5).
2. 200 mesh gold QUANTIFOIL R 2/2 holey carbon grids. See
Note 1.
3. Glow Discharge System.
4. Glass slide and glass petri dish. Manufacturer and size of the
slide and dish do not matter, as long as the dish is large enough
to hold the slide.
5. 70% ethanol.
2.2 PDL Coating
1. Poly-D-Lysine solution.
2. 0.2 μm PES vacuum filter.
2.3 Neuronal Culture
1. Hippocampal Neurons. We use a Complete Kit for E18 Sprague Dawley rat hippocampus (BrainBits). See Note 2.
2. 35 mm glass bottom cell culture dishes. See Note 3.
3. Culture Media: We use NbActiv4 media (BrainBits) with 1%
Pen-Strep, but standard neurobasal media and other variants
have been used successfully (see Note 2).
4. Trypan Blue Solution, 0.4%.
2.4 Fiducial Gold
1. Lyophilized BSA.
2. 10 nm colloidal gold nanoparticles.
2.5 Vitrification
1. Whatman Grade 1 Qualitative Filter Paper; 55 mm circles.
2. Plunge Freezer. We use the Thermo Fisher Scientific Vitrobot
Mark IV, but any should be able to work, even completely
manual setups without strict environmental controls.
3. Ethane Canister.
4. Liquid Nitrogen.
5. Liquid Nitrogen dewars for transport and storage.
6. CryoEM grid storage boxes.
7. Long (>6 in.) pair of blunt tweezers.
Cryotomography of Neurons
27