Computational protocol: A System for Performing High Throughput Assays of Synaptic Function

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Protocol publication

[…] Primary rat neuronal cultures on coverslips were infected with the hSyn-SypHy-AAV at 7 DIV. At 21 DIV, neurons were fixed in 4% formaldehyde, permeabilized in 0.3% Triton X-100, and blocked in 0.1% Triton X-100 plus 10% normal goat serum (Sigma). Neurons were incubated with primary antibodies, mouse anti-synaptotagmin (1∶1000; Synaptic Systems) and rabbit anti-GFP (1∶1000; Abcam), overnight at 4°C, rinsed in PBS, and incubated in secondary antibodies, Alexa-Fluor488 conjugated goat anti-rabbit IgG (1∶1000; Invitrogen) and Alexa-Fluor555 conjugated goat anti-mouse IgG (1∶1000; Invitrogen). Neurons were rinsed, mounted onto glass slides using Fluoromount G (Southern Biotech), and visualized with a Zeiss Axiovert Z1 microscope using a 40X 1.3NA oil immersion objective lens. Colocalization analysis was performed using ImageJ. […]

Pipeline specifications

Software tools Coloc, ImageJ
Application Microscopic phenotype analysis
Organisms Rattus norvegicus
Diseases Hereditary Central Nervous System Demyelinating Diseases