Jun 22, 2023
Active zone protein clusters and dopamine axons in striatal slices
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Chuyu Chen 2023. Active zone protein clusters and dopamine axons in striatal slices. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4q21zvo5/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: June 22, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83889
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 detail the procedures for measuring the size and density of active zone proteins and axon terminals with Imaris software.
Visualization of active zoon proteins and dopamine axons
Visualization of active zoon proteins and dopamine axons
Stain and amplify fluorescent labeled axons with GFP, cherry, and bassoon antibodies.
Capture z-stack images with 100x oil lens (Nikon N-SIM Structured Illumination Super-Resolution Microscope), 4 μm (0.1 μm steps)
9 images per section and 5 sections per mouse are used for analysis.
Quantification and colocalization analysis with Imaris 10.0
Quantification and colocalization analysis with Imaris 10.0
Open the 3D-reconstructed .nd2 files and convert to .ims files
Initiate the surface creation with surface module
Axon terminal
Rename the surface module to GFP/mch.
Select object-object statistics for algorithm settings.
Select smooth and set the surface detail at 0.0643 μm. Background subtraction set the diameter of the largest sphere which fit into 0.421 μm
Threshold set to auto for minimal value and none for max value, adject the value manually if needed.
Select “number of voxels of Img=1” for surface filter and set the min value to auto, adject the value manually if needed
Active zone protein
Select classify surfaces and object-object statistics for algorithm settings.
Select smooth and set the surface detail at 0.06 μm. Background subtraction set the diameter of the largest sphere which fit into 0.15 μm
Threshold set to auto for minimal value and none for max value, adject the value manually if needed. Select split touching objects with intensity based and enable seep points diameter set to 0.1 μm
Filter seed points with “Quality” and set the min and max value with auto.
Filter surfaces with “volume” and set the min at 0.003 μm^3 and max at 0.04 μm^3.
Filter colocalized active protein and axon terminal by select “overlapped volume ratio to surface surface=GFP/mch” and set the min=1 (100% volume of active zone protein overlapped with anxon).
Duplicate selection to new surface for statistic data of active zone protein within axon.
Measure the axon length with filament module.
Skip automatic creation, edit manually to 100% capture the axon surface.
Select source channel to GFP or mcherry. And use “AutoPath” to trace the generated GFP/mch surface.
Export all the statistical data to Excel for further analysis with GraphPad Prism9.