Immunofluorescence Assay to Evaluate Effect of Target Proteins on Neurite Outgrowth
Trascrizione
Developing neurons extend cytoplasmic processes from their cell bodies by neurite outgrowth to develop dendrites and axons, which are essential for neuron-to-neuron communication.
To study the effect of a target protein on neurite outgrowth, take a multiwell plate with a poly-D-lysine-coated coverslip at its base. The coverslip's surface has an adherent culture of primary cortical neurons derived from the embryonic rat brain.
Treat the neurons with a mix containing two types of plasmids — an EGFP plasmid encoding enhanced green fluorescent protein and a second plasmid encoding the target protein in a suitable transfection reagent.
The transfection reagent facilitates the entry of the plasmids into the cells.
Post-transfection, both plasmids get expressed, producing green fluorescent proteins and target proteins. The expression of EGFP imparts green fluorescence to the co-transfected cells.
Target proteins with neurite outgrowth-stimulating potential initiate cellular signaling, which promotes cytoskeleton reorganization and leads to the development of neuronal projections and their extensions.
Aspirate the spent medium, and fix the cells with paraformaldehyde. Counterstain the cells using red fluorescence-tagged anti-target protein antibodies that specifically bind expressed target proteins and impart additional red fluorescence to the cells.
Remove the coverslip from the culture plate, and observe it under a fluorescence microscope.
Co-transfected cells exhibiting a dual-colored fluorescence signal show longer neuronal processes than cells lacking red fluorescence, confirming the target protein's stimulating effect on neurite outgrowth.
