Immunofluorescence Assay to Evaluate Effect of Target Proteins on Neurite Outgrowth
Transcrição
After two days at 37 degrees Celsius and 5% carbon dioxide, dilute EGFP and target protein plasmid DNAs and transfection reagent in two separate 1.5-milliliter tubes, and then, mix them together. Transfect the cells with EGFP construct by adding the transfection mix to the wells.
After 24 hours, wash the cells with 37 degrees Celsius PBS, and fix them with 4% paraformaldehyde in PBS for 10 minutes in the dark at room temperature.
At the end of the incubation, wash the fixed cells three times with fresh PBS per wash, and add a minimal volume of fluorescence mounting medium onto one microscope glass slide, per sample. Then, carefully transfer the coated coverslips onto the mounting medium, with the samples facing the glass slides.
To image the neurite growth, select the 40x objective on an epi-fluorescent microscope, and click "Start" to capture images from at least 40 intact EGFP-positive neurons per transfection.
When all of the new rights have been imaged, open the captured images in ImageJ with the NeuronJ plugin, and measure the length of the longest neurite of each neuron from the cell body to the tip of the growth cone. Then, analyze the data obtained with the software, to determine the effect of the targeted proteins on neurite growth.
