TITLE: Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes
AUTHORS: Prerana  Gogoi, Momoko Shiozaki, Eric Gouaux

[DESCRIPTION]
Ionotropic glutamate receptors (iGluRs) at postsynaptic terminals mediate the majority of fast excitatory neurotransmission in response to release of glutamate from the presynaptic terminal. Obtaining structural information on the molecular organization of iGluRs in their native environment, along with other signaling and scaffolding proteins in the postsynaptic density (PSD), and associated proteins on the presynaptic terminal, would enhance understanding of the molecular basis for excitatory synaptic transmission in normal and in disease states. Cryo-electron tomography (ET) studies of synaptosomes is one attractive vehicle by which to study iGluR-containing excitatory synapses. Here we describe a workflow for the preparation of glutamatergic synaptosomes for cryo-ET studies. We describe the utilization of fluorescent markers for the facile detection of the pre and postsynaptic terminals of glutamatergic synaptosomes using cryo-laser scanning confocal microscope (cryo-LSM). We further provide the details for preparation of lamellae, between ~100 to 200 nm thick, of glutamatergic synaptosomes using cryo-focused ion-beam (FIB) milling. We monitor the lamella preparation using a scanning electron microscope (SEM) and following lamella production, we identify regions for subsequent cryo-ET studies by confocal fluorescent imaging, exploiting the pre and postsynaptic fluorophores.

[STEPS]
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
1. Remove the whole brains of three mice (14-23 weeks old), immediately after being euthanized, and rinse the brains in ice-cold PBS buffer pH 7.4.

SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
2. Add 10 mL of ice-cold homogenization buffer (HB) to the freshly dissected brain (net weight of three mice brain: ~1.55 gm).
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
3. Prior to using a glass Teflon dounce homogenizer, rinse all its parts with deionized (DI) water followed by rinsing with the HB, and allow it to cool in ice for 10-15 min.
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
4. Use a loose-fitted hand-held 15 mL glass Teflon dounce homogenizer to initially prepare a crude brain lysate using 4-5 strokes while maintaining the homogenizer in ice.
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
5. Further homogenize the brain lysate using a motor-driven dounce homogenizer for another 5-6 strokes, with each stroke followed by ~30 sec incubation of the glass tube containing the brain lysate in ice.
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
6. Collect the homogenate in a sterile 15 mL falcon centrifuge tube and centrifuge at 1000g for 10 min at 4 ºC.
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
7. Gently decant the supernatant (S1) to a fresh 15 mL tube. The pellet formed is very soft, hence care should be taken not to mix it with the supernatant.
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
8. Centrifuge the supernatant (S1) for another 20 min at 13,000g at 4 ºC using high-speed polycarbonate round bottom centrifuge tubes in a SS-34 fixed angle rotor to obtain the pellet containing synaptosomes. 
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
9. Remove the supernatant and gently resuspend the pellet in 1.5-2 mL of HB. Do not vortex. 
SECTION: Preparation of crude synaptosomes from vGLUT1-mVenus knock-in mouse
10. Add 50-100 nM of 15F1 Fab-mCherry to the resuspended synaptosome homogenate and gently nutate it for 1 h at 4 ºC. 
SECTION: Preparation of sucrose/Ficoll/Percoll density gradient
11. Prepare the required volume and concentration of sucrose/Ficoll/Percoll gradient buffers as described in “Reagent Setup” section. 
SECTION: Preparation of sucrose/Ficoll/Percoll density gradient
12. Use a 5 mL pipette or a 14G dispensing needle attached to a 10 mL syringe to slowly create the sucrose or Ficoll density gradient layers in an open-top thin wall ultra-clear tube. For Percoll density gradient separation, use 2 mL each of the density gradient buffers to create the layers in a 10.4 mL polycarbonate bottle with cap assembly. 
SECTION: Preparation of sucrose/Ficoll/Percoll density gradient
13. Start from the heaviest density to the lightest, working the way up from bottom to top. For optimal results, hold the tube at 45 º angle and maintain the flow as one drop at a time out of the pipette tip or needle, while creating a gentle stream of the buffer trickling down the walls of the tube. 
SECTION: Preparation of sucrose/Ficoll/Percoll density gradient
14. After preparation of each layer, let the tube stand for ~2 min before layering the next gradient on top. 
SECTION: Preparation of sucrose/Ficoll/Percoll density gradient
15. Once all the gradient layers are being formed, allow the tubes to sit in ice or 4 ºC for 10-15 min.
SECTION: Preparation of sucrose/Ficoll/Percoll density gradient
16. Layer the synaptosome homogenate on top of the density gradient layers and let sit for ~2 mins.
SECTION: Density gradient centrifugation
17. For sucrose and Ficoll density gradient centrifugation, spin the tubes at 30,965g for 70 min at 4 ºC using a swinging bucket rotor (SW 27) in an Optima L-80 XP ultracentrifuge, Beckman Coulter. For Percoll density gradient centrifugation, use a fixed bucket rotor (Type 75 Ti in an Optima L-80 XP ultracentrifuge, Beckman Coulter) to centrifuge at 12,854g for 15 min at 4 ºC. It is important to set the acceleration and deceleration speed to 6 and 4, respectively, in order to avoid disturbing the separated fractions and gradient layers.
SECTION: Retrieval of the enriched synaptosomes
18. Enriched synaptosomes form a visibly distinct layer in between 0.8 and 1.2 M sucrose, 8 and 14% Ficoll and 15 and 23% Percoll (S1 Fig.). In the cases of sucrose and Ficoll, wherein a thin-walled tube is used, use an 18-gauge needle attached to a 10 mL syringe to access the synaptosome band by rupturing the side of the tube. 
SECTION: Retrieval of the enriched synaptosomes
19. An alternative method to retrieve the synaptosomes prepared using the Percoll density gradient is to use the 14G dispensing needle attached to a 10 mL syringe. Gently remove the unwanted layers from the top until the synaptosome band can be accessed. In all instances, care should be taken not to disturb the gradient layers.
SECTION: Transferring the synaptosomes to an isotonic buffer
20. To remove sucrose, Ficoll or Percoll after density gradient centrifugation, dilute the retrieved synaptosomes to at least five-fold in HB and centrifuge at 30, 000g for 15 min at 4 ºC using a 70 mL polycarbonate bottle assembly in a Type 45 Ti fixed angle rotor compatible with Optima L-80 XP ultracentrifuge, Beckman Coulter.
SECTION: Transferring the synaptosomes to an isotonic buffer
21. Remove the supernatant and resuspend the synaptosomes in PBS pH 7.4, or any desired buffer.
SECTION: Transferring the synaptosomes to an isotonic buffer
22. Add 5 mL PBS pH 7.5 to the synaptosomes and gently resuspend the pellet using a pipette without causing froth formation. Sufficient resuspension is critical for well-dispersed non-aggregated synaptosomes.
SECTION: Transferring the synaptosomes to an isotonic buffer
23. Immediately proceed to cryo-EM grid preparation because synaptosomes stored on ice or at 4 °C tend to aggregate.
SECTION: Filtration of synaptosomes using a thermobarrel extruder
24. Prepare the synaptosomes as described in steps 1-19. 
SECTION: Filtration of synaptosomes using a thermobarrel extruder
25. Resuspend the synaptosomes in 10 mL PBS pH 7.4.
SECTION: Filtration of synaptosomes using a thermobarrel extruder
26. Set up the extruder as described in manufacturer’s manual. 
SECTION: Filtration of synaptosomes using a thermobarrel extruder
27. Use a glass Pasteur pipet to carefully apply 0.5-1 mL of the synaptosomes on top of the 1 µm filter paper in the extruder and continue for the entire volume.
SECTION: Filtration of synaptosomes using a thermobarrel extruder
28. Carefully collect the filtered synaptosomes in a clean 50 mL Falcon tube placed in ice. 
SECTION: Filtration of synaptosomes using a thermobarrel extruder
29. Repeat the filtration for nine passes, each time using the filtered synaptosomes obtained from the preceding step. 
SECTION: Filtration of synaptosomes using a thermobarrel extruder
30. Replace the filter after passing 3-5 mL of the synaptosomes to avoid clogging up the extruder.
SECTION: FSEC analysis of glutamatergic synaptosomes
31. Before subjecting the retrieved glutamatergic synaptosome sample to FSEC analysis, solubilize the synaptosomes using digitonin containing solubilization buffer as described in steps 32-36.
SECTION: FSEC analysis of glutamatergic synaptosomes
32. Take 0.5-1 mL of synaptosomes and centrifuge at 15, 000g for 10 min. 
SECTION: FSEC analysis of glutamatergic synaptosomes
33. Remove the supernatant and resuspend the synaptosomes in 100 µL of TBS pH 8 buffer.
SECTION: FSEC analysis of glutamatergic synaptosomes
34. Add 100 µL of solubilization buffer (refer “Reagent set up”) to the homogeneously resuspended synaptosomes.
SECTION: FSEC analysis of glutamatergic synaptosomes
35. Gently nutate the samples for 1 h at 4 ºC.
SECTION: FSEC analysis of glutamatergic synaptosomes
36. Centrifuge the solubilized synaptosomes at 70,000g for 40 min at 4 ºC.
SECTION: FSEC analysis of glutamatergic synaptosomes
37. Collect the supernatant and analyze 70 µL by FSEC.
SECTION: FSEC analysis of glutamatergic synaptosomes
38. Monitor for the presence of vGLUT1-mVenus and AMPAR-15F1 Fab-mCherry signals using mVenus (λex: 510 nm, λem: 535 nm) and mCherry (λex: 580 nm, λem: 610 nm) channels, respectively.
SECTION: Preparation of cryo-EM grids using a manual plunger
39. Prior to cryo-EM grid preparation, add 2 µL of 10 nm gold fiducialto 10 µL synaptosomes and mix well.
SECTION: Preparation of cryo-EM grids using a manual plunger
40. Plunge-freeze the synaptosomes using a manual plunge freezer. Apply 2.5 µL of synaptosomes to the glow-discharged side of the grid and blot away the excess liquid from the back of the grid using blotting paper.
SECTION: Preparation of cryo-EM grids using a manual plunger
41. After blotting, immediately vitrify the grid in propane/ethane 50%/50% (v/v) mixture, cooled liquid nitrogen. Carefully transfer grids to a grid box placed in liquid nitrogen.
SECTION: Sample clipping
42. Mark the cryo-FIB-AutoGrids with an alcohol-resistant marker on the single embedded mark 90 º clockwise from the milling slot. This marking will facilitate the correct alignment of the grid in the cryo-FIB shuttle and later in the transmission electron microscope (TEM) cassette for cryo-ET.
SECTION: Sample clipping
43. Cool down the transfer station with liquid nitrogen and place the marked cryo-FIB-AutoGrid upside down in the grid mounting position.
SECTION: Sample clipping
44. Place the grid on top of the cryo-FIB-AutoGrid with the sample side of the grid facing downward.
SECTION: Sample clipping
45. Load a C-ring into the tip of a C-ring insertion tool using a tweezer and gently press the C-ring on a flat and firm surface to align it with the rim of the insertion tool.
SECTION: Sample clipping
46. Precool the C-ring loaded insertion tool in liquid nitrogenand gently push to the C-ring to clip the sample cryo-EM grid onto the cryo-FIB-AutoGrid while holding the C-ring insertion tool vertically. 
SECTION: Sample clipping
47. Use a blunt-end tweezer to gently turn the clipped cryo-FIB AutoGrid upside down to ensure that the grid is clipped correctly.
SECTION: Imaging glutamatergic synaptosomes using cryo-LSM
48. For imaging the glutamatergic synaptosomes on the cryo-EM grids, use a fluorescence microscope equipped with a cryo-stage. Here we used a Zeiss LSM 980 with Airyscan 2 coupled with a Linkam cryo-stage. 
SECTION: Imaging glutamatergic synaptosomes using cryo-LSM
49. Carefully place the clipped cryo-EM grid in the Cassette Transport in the liquid nitrogen-filled Linkam cryo-stage.
SECTION: Imaging glutamatergic synaptosomes using cryo-LSM
50. Launch the ZEN software.
SECTION: Imaging glutamatergic synaptosomes using cryo-LSM
51. Turn on the lasers, select “Acquisition” from the main toolbar and acquire the image using the WF for the low magnification image of the grid using the 5X objective lens.
SECTION: Imaging glutamatergic synaptosomes using cryo-LSM
52. Change to the 10X objective lens and acquire a medium magnification image of the grid. At medium magnification, one-fourth of the grid can be imaged at one time. Acquire images of the areas which have the least number of broken squares. If imaging for cryo-FIB milling, it is important to obtain an image of the mid area of the grid, as this area is often the best accessible by the ion beam during cryo-FIB milling.
SECTION: Imaging glutamatergic synaptosomes using cryo-LSM
53. Change to the 100X objective lens. Precaution should be taken to first correctly focus the grid at 5X and 10X, so as to avoid the crashing of the objective lens of the microscope into the Linkam cryo-stage. 
SECTION: Imaging glutamatergic synaptosomes using cryo-LSM
54. While using the 100X objective lens, set the Airyscan light path configuration to confocal image acquisition. 
SECTION: Imaging glutamatergic synaptosomes using cryo-LSM
55. Acquire images at the desired areas and save images as .tif or .jpg files for later use during alignment in cryo-FIB-SEM.
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
56. Load the cryo-FIB-AutoGrid into the 45º pre tilited cryo-FIB-SEM shuttle. Make sure the milling slot of the cryo-FIB-Autogrid is aligned to the vertical position. Load the shuttle into the cryo-FIB-SEM chamber by using the transfer rod. 
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
57. Using Maps software, acquire SEM tile set image of the grid with 550 µm horizontal field width (HFW) and 3072 x 2048 pixel (px) or higher to clearly visualize surface structures. Import fluorescent light microscope (FLM) image into Maps software and align to the SEM tile set image. There will be no apparently visible features corresponding to synaptosomes during SEM imaging. Hence, ensure that the FLM images are very well aligned to the SEM image by checking features such as the center of the grid, grid bars or broken carbon films for optimal location of the target.
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
58. Within the Maps software, pick the lamella sites close to the center of the grid squares based on the FLM image. During a session of 6-8 hrs as many as 10 lamellae can be prepared.
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
59. Sputter coat the surface of the grid with a conductive layer of platinum (Pt) for 10 sec at 30 mA and 10 Pa, followed by deposition of an additional Pt gas injection system (GIS) layer for another 15 secs to protect the lamella surface and prevent the curtaining effect.
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
60. Use AutoTEM 2.0 software for milling. Set up the milling parameters as shown in S1 Table. Parameters such as milling current and depth correction need to be adjusted according to the ice thickness of the lamella sites. Final thickness is set to 350 nm so each lamella can be further milled down to 100-200 nm by manual milling. 

 
 
       Parameters            Milling angle (º)         12            Size (µm)         12x 5            Final thickness (nm)         350            Correction factor (%)         0.6                      lamella thickness (Pattern offset)   (µm)         Milling current (nA)         Depth correction (%)         Width overlap (Front and rear)   (µm)            Rough milling         1         1         70         400            Medium milling         0.75         0.3         70         300            Fine milling         0.3         0.1         70         200            Finer milling         0.15         0.05         70         100            Polishing         0.025         0.03         80         N/A      

 
 
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
61. In the preparation step of AutoTEM 2.0, perform eucentric tilting with a 10 º tilt step followed by lamella placement to register final lamella position. 
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
62. In the milling step from rough Milling to finer milling, use stepwise mode to thin down each lamella site. Mill micro-expansion joints, also known as stress relief cuts, alongside the lamella to avoid lamella bending [58]. After starting the milling process, make sure the parameters are suitable for the sample and adjust the milling current or depth correction as required. The milling step would render a lamella with a thickness of ~600nm. 
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
63. Further mill down the lamella to ~350 nm during the polishing step. 
SECTION: Cryo-FIB milling of glutamatergic synaptosomes
64. To obtain the final lamella within the thickness range of ~100-200 nm, manually polish using 10 or 30 pA milling current at 30 kV. Use rectangle pattern to visually inspect milled materials during thinning. 
SECTION: Imaging on cryo-FIB milled lamella
65. To ensure the presence of glutamatergic synaptosomes, subject the FIB-milled lamellae to cryo-LSM imaging.
SECTION: Imaging on cryo-FIB milled lamella
66. Repeat steps 48-55 for imaging.