Mar 07, 2025
Striatal synaptosome preparation
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Giulia Tombesi, Loukia Parisiadou 2025. Striatal synaptosome preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l6dxmrvqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 06, 2024
Last Modified: March 07, 2025
Protocol Integer ID: 114372
Keywords: ASAPCRN
Funders Acknowledgements:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
Abstract
This protocol describes how to isolate striatal synaptosomes form brain tissue and process them for imaging purposes.
Before start
Prepare the following solutions:
- Homogenizing buffer: 4 millimolar (mM) HEPES, 340 millimolar (mM) sucrose and 1x protease inhibitor cocktail
- Sucrose solution 0.8 Molarity (M) + 1x protease inhibitor cocktail
- Sucrose solution 1.2 Molarity (M) + 1x protease inhibitor cocktail
Striatal synaptosome preparation
Striatal synaptosome preparation
1h 35m
1h 35m
Homogenize one Striatum by 12 strokes with a glass tissue grinder in 1 mL of homogenizing buffer
Centrifuge the homogenate at 1000 x g for 00:10:00 at 4 °C
10m
Centrifuge the supernatant at 12500 x g for 00:15:00 at 4 °C
15m
Discard the supernatant and retain the pellet
Resuspend the pellet in 1 mL of homogenizing buffer and perform 6 strokes in the glass tissue grinder
Add 1 mL of homogenate to the top of a sucrose gradient made as follow:
- 5 mL of 1.2 Molarity (M) sucrose
- 5 mL of 0.8 Molarity (M) sucrose
Centrifuge the homogenate at 23600 x g for 01:10:00 at 4 °C
1h 10m
Carefully collect the syanptosome layer from the interface of the two sucrose layers using a 1ml syringe (aspirate up 1ml)
Striatal synaptosomes plating for imaging
Striatal synaptosomes plating for imaging
40m
40m
Dilute synaptosomes in 1 Molarity (M) sucrose to the desired concentration and plate them on poly-L-lysine-coated #1.5 coverslips in a 24-well plate
Centrifuge the plate at 4000 rpm for 00:30:00 at 4 °C to promote adhesion
30m
Fix the synaptosomes with 4% PFA for 00:10:00 at Room temperature
10m
Striatal synaptosomes staining
Striatal synaptosomes staining
2h 55m
2h 55m
Saturate synaptosomes in 5 % (v/v) donkey serum in PBS for 01:00:00
1h
Permeabilize synaptosomes in 0.5 % (v/v) Triton X-100 in PBS for 00:10:00
10m
Incubate synaptosomes with primary antibodies in 0.1 % (v/v) Triton X-100, 1 % (v/v) donkey serum PBS Overnight at 4 °C
10m
Perform three 00:05:00 washes
5m
Incubate synaptosomes with the secondary antibodies in 0.1 % (v/v) Triton X-100, 1 % (v/v) donkey serum PBS for 01:30:00
1h 30m
Mount synaptosomes onto slides
Striatal synaptosomes imaging and analysis (example)
Striatal synaptosomes imaging and analysis (example)
Acquire a single optical section for each area of syanptosomes with a confocal microscope system
Perform image analysis with ImageJ software
Regarding intensity analysis, background was subtracted from all channels using the “rolling ball” ImageJ plugin with a radius of 33.0 pixels. Automatic segmentation (threshold: default; analyze particles: size 0.04-0.40; circularity: 0.30-1.00) was exploited to define ROIs in VAMP2 channel. The intensity of the signal was measured within individual ROIs and only the ROIs positive for VAMP2 were kept. As a rule, a ROI is considered to contain the target protein when the average fluoresce intensity of that protein within the ROI is more than 2 times the average intensity of all pixels in the image and is considered as not containing the target protein when the intensity of the target protein within the ROI is below 2 times the average intensity of all pixels. To identify VAMP2 containing synaptosomes that are also positive for TH, the intensity of TH signal was measured within VAMP2 positive ROIs. The intensity of the protein of interest signal (e.g. BSN, RIM) was measured in the double-positive ROIs to keep only the triple-positive ROIs and the mean intensity value of the protein of interest was calculated and plotted.
Regarding BSN area, automatic segmentation was used to define the ROIs set both for BSN and TH (threshold: default; analyze particles: size 0.04-0.40 for BSN and size 0,06-0,60 for TH; circularity: 0.30-1.00). BSN and TH ROIs sets were used to define the overlapped ROIs exploiting “AND” function in the ROI Manager. The intensity of VAMP2 signal was used to define the BSN-TH overlapped ROIs positive for VAMP2. Finally, the average area value of BSN localized within TH and VAMP2 positive synaptosomes was determined using the “set measurement” and “mesure” ImageJ functions.
Regarding BSN %, automatic segmentation was used to define ROIs in TH channel (threshold: default; size 0,06-0,60; circularity: 0.30-1.00). TH ROIs positive for VAMP2 were defined measuring the intensity signal of VAMP2. BSN intensity signal was then measured within the double positive ROIs to obtain the number of ROIs that were triple positive. Automatic segmentation was used to define the total number of ROIs in BSN channel (threshold: default; size 0,04-0,40; circularity: 0.30-1.00). Finally, the % of BSN positive synaptosome localizing with VAMP2 and TH double positive synaptosomes was calculated.