Materials and reagents

Biological materials

1. 10-week-old Sprague-Dawley rats of either sex (Charles River Laboratories)

Reagents

1. Isoflurane (Piramal, catalog number: NDC 66794-017-25)

2. Sucrose (Sigma-Aldrich, catalog number: S9378)

3. HEPES (Fisher BioReagent, catalog number: BP310-1)

4. EDTA (Millipore, catalog number: 324503)

5. 10-nm BSA-gold beads (Aurion, catalog number: 25486)

6. NaOH (Fisher, catalog number: AC259860010)

7. Ethane gas (A-L Compressed Gases, catalog number: AGETHANECP-7)

8. Liquid nitrogen (A-L Compressed Gases, catalog number: GSINL240)

Solutions

1. 0.5 M EDTA stock solution (see Recipes)

2. 10-nm gold beads solution (see Recipes)

3. Iso-osmotic homogenization buffer (see Recipes)

Recipes

1. 0.5 M EDTA stock solution

To prepare 0.5 M EDTA (pH 8.0), dissolve 93 g of EDTA disodium salt in ~400 mL of deionized water in a 500 mL autoclavable screw-cap bottle with a magnetic stir bar. The EDTA will not dissolve until the pH is adjusted. Slowly add ~10 g of NaOH pellets while stirring until the pH reaches 8.0. Once fully dissolved, transfer the solution to a 500 mL graduated cylinder, adjust to the final volume with deionized water, remove the stir bar, and return the solution to the bottle. Autoclave and store at room temperature.

2. 10-nm gold beads solution

To prepare a 10 nm BSA-gold solution, centrifuge 300 μL of BSA-conjugated gold nanoparticles at 161,000× g for 30 min at 4 °C. Carefully discard the supernatant without disturbing the pellet. Resuspend the pellet gently in 150 μL of iso-osmotic homogenization buffer (Recipe 3) by pipetting or brief vortexing. Store the solution at 4 °C and use within a week. 10-nm gold beads are added to the sample when plunge freezing. These gold beads will be used as fiducial markers to align the tilt series during reconstruction.

3. Iso-osmotic homogenization buffer

Prepare 500 mL of homogenization buffer as a 0.32 M sucrose solution by adding 54.77 g of sucrose in ~400 mL of deionized water and stirring in a 500 mL autoclavable screw-cap bottle with a magnetic stir bar. This is then supplemented with 0.477 g of HEPES and 1 mL of 0.5 M EDTA stock (Recipe 1) while stirring until dissolved, and finally adjusted to pH 7.4.

Laboratory supplies

1. Quantifoil R2/2 Cu 200 mesh EM grids (Quantifoil)

2. Cryo grid box (SubAngstrom, catalog number: SBV01)

Equipment

1. Small animal decapitator (Fisher Scientific, catalog number: 10-000-124)

2. Overhead stirrer (Electron Microscopy Sciences, catalog number: 6480610)

3. 10-mL Teflon head tissue grinder (Electron Microscopy Sciences, catalog number: 6479310)

4. Centrifuge (Eppendorf, model: 5415D)

5. FEI Vitrobot Mark III (FEI, now Thermo Fisher Scientific)

6. Titan Krios G4 Cryo-EM (Thermo Fisher Scientific)

7. K3 camera (Gatan)

Software and datasets

1. Thermo Fisher Tomography 5 https://www.thermofisher.com/us/en/home/electron-microscopy/products/software-em-3d-vis/tomography-software.html

2. IMOD https://bio3d.colorado.edu/imod/ [6]

3. MotionCorr2 https://emcore.ucsf.edu/ucsf-software [7]

4. Gctf https://github.com/JackZhang-Lab/GCTF [8]

Procedure