Computational protocol: Proteomic Studies of a Single CNS Synapse Type: The Parallel Fiber/Purkinje Cell Synapse

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Protocol publication

[…] The MRCKγ cDNA was amplified from cerebellar cDNA. The MRCKγDN construct was obtained by deleting the sequence encoding for amino acids 1–426, and by replacing it with ATG. MRCKγ and eGFP cDNAs were subcloned in the bidirectional Tet-responsive vector pBI (Clontech).Primary neuronal cultures were prepared from E15 mouse embryos (Swiss strain). Cortices were dissected and triturated using a fire-polished Pasteur pipette and 0.05% trypsin. Neurons were plated on poly-d-lysine and laminin-coated coverslips at a density of 1.5 × 105 cells/cm2 and cultured in neurobasal medium supplemented with 2% B27 supplement, 0.5 mM glutamine, and antibiotics. Transfections were performed at DIV7 with a 1:1 ratio of a tTA-expressing plasmid and the bidirectional vector containing GFP (with or without the kinases) using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen).Dendrites of transfected neurons were imaged using a confocal microscope and a 63× objective with a 5× zoom. Quantifications of protrusion density and length were performed using the NeuronJ plugin and the ImageJ software on several dendrites per neuron (at least five different cells per transfection condition, four independent experiments). A total of 1,029, 861, and 950 protrusions were counted and measured for the GFP, DN, and FL transfections, respectively. Statistical analysis was performed using the GraphPad Prism software. […]

Pipeline specifications

Software tools NeuronJ, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Mus musculus