Isolation, cryo-laser scanning confocal microscope imaging and cryo-FIB milling of mouse glutamatergic synaptosomes

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.

Add 10 mL of ice-cold homogenization buffer (HB) to the freshly dissected brain (net weight of three mice brain: ~1.55 gm).

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.

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.

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.

Collect the homogenate in a sterile 15 mL falcon centrifuge tube and centrifuge at 1000 g for 10 min at 4 ºC.

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.

Centrifuge the supernatant (S1) for another 20 min at 13,000 g at 4 ºC using high-speed polycarbonate round bottom centrifuge tubes in a SS-34 fixed angle rotor to obtain the pellet containing synaptosomes.

Remove the supernatant and gently resuspend the pellet in 1.5-2 mL of HB. Do not vortex.

Add 50-100 nM of 15F1 Fab-mCherry to the resuspended synaptosome homogenate and gently nutate it for 1 h at 4 ºC.

Prepare the required volume and concentration of sucrose/Ficoll/Percoll gradient buffers as described in “Reagent Setup” section.

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.

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.

After preparation of each layer, let the tube stand for ~2 min before layering the next gradient on top.

Once all the gradient layers are being formed, allow the tubes to sit in ice or 4 ºC for 10-15 min.

Layer the synaptosome homogenate on top of the density gradient layers and let sit for ~2 mins.

For sucrose and Ficoll density gradient centrifugation, spin the tubes at 30,965 g 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 12,854 g 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.

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.

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.

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, 000 g 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.

Remove the supernatant and resuspend the synaptosomes in PBS pH 7.4, or any desired buffer.

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.

Immediately proceed to cryo-EM grid preparation because synaptosomes stored on ice or at 4 °C tend to aggregate.

Prepare the synaptosomes as described in steps 1-19.

Resuspend the synaptosomes in 10 mL PBS pH 7.4.

Set up the extruder as described in manufacturer’s manual.

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.

Carefully collect the filtered synaptosomes in a clean 50 mL Falcon tube placed in ice.

Repeat the filtration for nine passes, each time using the filtered synaptosomes obtained from the preceding step.

Replace the filter after passing 3-5 mL of the synaptosomes to avoid clogging up the extruder.

Before subjecting the retrieved glutamatergic synaptosome sample to FSEC analysis, solubilize the synaptosomes using digitonin containing solubilization buffer as described in steps 32-36.

Take 0.5-1 mL of synaptosomes and centrifuge at 15, 000 g for 10 min.

Remove the supernatant and resuspend the synaptosomes in 100 µL of TBS pH 8 buffer.

Add 100 µL of solubilization buffer (refer “Reagent set up”) to the homogeneously resuspended synaptosomes.

Gently nutate the samples for 1 h at 4 ºC.

Centrifuge the solubilized synaptosomes at 70,000 g for 40 min at 4 ºC.

Collect the supernatant and analyze 70 µL by FSEC.

Monitor for the presence of vGLUT1-mVenus and AMPAR-15F1 Fab-mCherry signals using mVenus (λ_ex: 510 nm, λ_em: 535 nm) and mCherry (λ_ex: 580 nm, λ_em: 610 nm) channels, respectively.

Prior to cryo-EM grid preparation, add 2 µL of 10 nm gold fiducialto 10 µL synaptosomes and mix well.

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.

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.

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.

Cool down the transfer station with liquid nitrogen and place the marked cryo-FIB-AutoGrid upside down in the grid mounting position.

Place the grid on top of the cryo-FIB-AutoGrid with the sample side of the grid facing downward.

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.

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.

Use a blunt-end tweezer to gently turn the clipped cryo-FIB AutoGrid upside down to ensure that the grid is clipped correctly.

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.

Carefully place the clipped cryo-EM grid in the Cassette Transport in the liquid nitrogen-filled Linkam cryo-stage.

Launch the ZEN software.

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.

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.

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.

While using the 100X objective lens, set the Airyscan light path configuration to confocal image acquisition.

Acquire images at the desired areas and save images as .tif or .jpg files for later use during alignment in cryo-FIB-SEM.

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.

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.

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.

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.

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.

A	B	C	D	E
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

S1 Table: Conditions for cryo-FIB milling of synaptosomes

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.

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.

Further mill down the lamella to ~350 nm during the polishing step.

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.

To ensure the presence of glutamatergic synaptosomes, subject the FIB-milled lamellae to cryo-LSM imaging.

Repeat steps 48-55 for imaging.