Adult mouse brain: All vGLUT1-mVenus knock-in mice (both male and female) were euthanized under Institutional Animal Care and Use Committee (IACUC) protocols, consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association (AVMA) and carried out only by members of Dr. Gouaux’s lab approved on the IACUC protocol TR01_IP00000905.
Sucrose (Sigma, cat. no.84097)
Ficoll 400 (Sigma, cat. no. F2637)
Percoll (GE Healthcare, cat. no. 17-0891-02)
HEPES (Fisher Bioreagents, cat. no. BP310-1)
Leupeptin (Sigma, cat. no. L0649)
Aprotinin (Sigma, cat. no. A1153)
Pepstatin A (Sigma, cat. no. P4265)
Gold fiducial beads, 10 nm (Ted-Pella, Inc., 90010-5)
50%/50% ethane and propane mixture
Digitonin (Apollo Scientific, cat. no. 11024-24-1)
Dissection tools
18-G needle (18G x 1 ½) (BD Precision Glide, Cat. no. 305196)
Dispensing needle, 14G (C-U Innovations)
Glass Teflon dounce homogenizer
38.5 mL, open-top thinwall ultra-clear tube, 25 x 89mm (Beckman Coulter, cat. no. 344058)
Nalgene™ high-speed polycarbonate round bottom centrifuge tubes (Thermo scientific, cat. no. 3117-0120)
10.4 mL, polycarbonate bottle with cap assembly, 16 x 76mm (Beckman Coulter, cat. no. 355603)
SS-34 fixed angle rotor (Sorvall)
Type 75 Ti fixed angle rotor (Beckman Coulter)
Type 45 Ti fixed angle rotor (Beckman Coulter)
SW27 swinging bucket rotor (Beckman Coulter)
RC5C refrigerated floor centrifuge (Sorvall)
Optima L-80 XP ultracentrifuge (Beckman Coulter)
Optima MAX-XP ultracentrifuge (Beckman Coulter)
Zeiss LSM 980 with Airyscan 2 (Zeiss)
Cryo-correlative microscopy stage (Linkam, cat. no. CMS196V)
Aquilos 2 Cryo-FIB (Thermo Scientific)
R 2/1 Holey Carbon-coated 200-mesh gold grids (Quantifoil Micro Tools GmbH, cat. no. Q51340)
Blotting paper (Ted pella, Inc., cat. no. 47000)
Steriflip-GP sterile centrifuge tube top filter unit (Millipore, cat. no. SCGP00525)
70 mL, Polycarbonate bottle assembly, 38 x 102 mm (Beckman Coulter, cat. no. 355622)
Autogrid or C-Clip ring (Thermo Scientific, cat. no. 1036173)
C-Clip (Thermo Scientific, cat. no. 1036171)
AutoGrid container (Thermo Scientific, cat. no. 1084591)
Thermobarrel extruder (Lipex, Transferra Nanosciences Inc.)
Cytiva Whatman nucleopore hydrophilic membrane, 1 µm (Fisher, cat. no. 18400175)
Fluorescence-detection size-exclusion chromatography (FSEC) [56].
The volume of reagents used in this protocol is optimized for synaptosome preparation from three adult whole mouse brains (each brain ~0.5 gm)
Homogenization buffer (HB)
Freshly prepare 50 mL homogenization buffer (HB) by mixing 10 mM HEPES pH 7.4 and 0.32 M sucrose. Add protease inhibitors to final concentrations of 0.8 µM aprotinin, 2 µg/mL leupeptin and 2 µM pepstatin A to the HB. Filter the HB using a Steriflip-GP sterile centrifuge tube top filter unit (0.22 µm) and store in ice until use.
Sucrose gradient buffer: Prepare 10 mL each of 0.8 and 1.2 M sucrose in HB.
Ficoll gradient buffer: Prepare 10 mL each of 8 and 14% (w/v) Ficoll in HB. Percoll gradient buffer: Prepare 5 mL each of 3, 10, 15 and 23% (v/v) Percoll in HB.
Tris-buffered saline (TBS)
Mix 20 mM Tris HCl pH 8.0 and 150 mM NaCl; filter the buffer using 0.2-µm filter and store at room temperature (RT; 25 ºC).
Freshly prepare solubilization buffer by mixing 20 mM Tris HCl pH 8.0, 150 mM NaCl and 2% (w/v) digitonin. Allow the digitonin to dissolve into the buffer solution by placing it on a nutator overnight at 4 ºC. The digitonin containing buffer will appear cloudy and can be used directly without filtration at this stage. Immediately before use, add aprotinin, leupeptin and pepstatin A to final concentrations of 0.8 µM, 2 µg/mL and 2 µM, respectively.
Mix 20 mM Tris HCl pH 8, 150 mM NaCl and 0.075% (w/v) digitonin. Allow digitonin to dissolve into the buffer solution by placing on a magnetic stirrer overnight at 4 ºC. Centrifuge at 9,700g for 20 min at 4 ºC to remove residual digitonin followed by filtering the buffer using a 0.2-μm filter. Store the buffer at 4 °C for up to one week and filter each time before use.
Centrifuge 500 µL of the 10 nm gold fiducial solution at 21,130g for 30 min in a benchtop centrifuge. Remove the supernatant without disturbing the soft pellet of the gold beads using a pipette. Add 500 µL phosphate-buffered saline (PBS) pH 7.4 buffer to wash the gold beads and subject it to another round of centrifugation at 21,130g for 30 min followed by removing the supernatant using a pipette. Prepare the final stock of the gold fiducial beads at an optical density (OD) of 15-10 by adding PBS pH 7.4 to make the final volume of 20 µL.
The success of the density gradient separation of synaptosomes is critically dependent on the centrifuge, rotors and tubes used. Thus, care must be taken to use the same, or similar, rotors and centrifugation conditions mentioned in this protocol while preparing the synaptosomes. Pre cool all the rotors (SS-34, Ti-45, Ti-75 and SW-28) used to prepare the synaptosome samples to 4 ºC.
Zeiss cryo-laser scanning confocal microscope (cryo-LSM 980) and Linkam cryo-stage
Turn on the HXP lamp 15-30 min prior to use for the fluorescent lamps to reach full brightness. Simultaneously, turn on the component, the PC connected to the microscope and launch the ZEN software.
To connect the Linkam stage to the microscope, launch the MTB configuration and set “Linkam” as the active configuration. Place the desired cassette transporter (single or multi-port) in the cassette prior to cooling down the cryo-stage. Connect the Linkam cryo-stage to the microscope and allow liquid nitrogen from the dewar connected to the cryo-stage to flow and cool down the Linkam chamber and stage to -196 ºC. The temperature of the dewar, chamber and stage must be constantly maintained at -196 ºC while the microscope is in use.
Set up wide field (WF): Set the Track1, Track2 and Track3 as bright field, mVenus (laser: 514 nm) and mCherry (laser: 561 nm) channels, respectively.
Set up Airyscan: Set Track1 to Ch2 for the Airyscan detector and the remaining two tracks as mVenus (laser: 514 nm) and mCherry (laser: 561 nm) channels .
Set the Pinhole and master gain at 184 µm and 500-800 V, respectively.
To obtain the confocal image of a complete grid square, set the crop area to 0.6x. To obtain a higher signal-to-noise ratio, set the pixel time 4-6, bidirectional scanning, with an averaging of 2x. These parameters should be adjusted according to specific requirements.
Fluorescence-detection size-exclusion chromatography (FSEC)
Load the analyte onto a Superose 6 column (10/30, Amersham Biosciences) that has been preequilibrated with FSEC buffer. The eluent from the size exclusion chromatography (SEC) column is passed through Shimadzu fluorometer (RF-20Axs) fitted with flow cells, as described in manufacturer’s instructions. The fluorometer settings are as follows: band-pass, 3 nm/3 nm; λex: 510 nm (mVenus) and 580 (mCherry), λem: 535 nm (mVenus) and 610 (mCherry); time increment, 1 s; integration time, 1 s; and recording time, 3,000–3,600 s. During the run, the flowrate for separation was maintained at 0.5 mL/min. A detailed description of FSEC can be found in [56].
Aquilos 2 cryo-focused ion beam-scanning electron microscope (cryo-FIB-SEM)
Purge the cooling line by flowing liquid nitrogen at 190 mg/s for at least 30 min. Purge argon lines by two purging cycles. If the shuttle is on the stage inside the chamber, remove it using a transfer rod and place it in the preparation box. Purge gas injection system (GIS) at room temperature for 30-60 seconds. Fill the heat exchanger dewar with liquid nitrogen and cool down the system by inserting the heat exchanger in the dewar. The temperature of the stage and cryo shield should be below -185 ºC and -182 ºC, respectively.