Immunofluorescence Assay to Evaluate Effect of Target Proteins on Neurite Outgrowth

1. Preparation of Coverslips

1. Place a sterile 18 mm circular coverslip into each well of a 12-well tissue culture plate.
2. Coat the coverslip with 5 µg/mL poly-D-lysine solution in a humidified 37 °C incubator for at least 1 hr.
3. Aspirate the poly-D-lysine solution from the tissue culture plate and rinse the coated coverslips once with sterile water.

2. Cell Transfection and Fixation

NOTE: All procedures in step 2 are performed inside a Class II Biosafety cabinet.

1. Transfect 0.5 µg of EGFP construct (pEGFP-C1) to primary cortical neurons together either with or without 0.5 µg of POI by using 1 µL of transfection reagent (e.g., Lipofectamine 2000). Use the manufacturer's instructions.
   NOTE: Mammalian expression constructs were prepared by using an endotoxin-free plasmid preparation kit. Treatment with chemicals/molecules (in this manuscript cytochalasin D (Cyto D) and nerve growth factor (NGF) were used) can be done at 6 hr after transfection.
2. Aspirate the culture medium 24 h post-transfection and wash the transfected cells once with 37 °C PBS (10 mM sodium phosphates, 2.68 mM potassium chloride, 140 mM sodium chloride).
3. Fix the cells with 4% paraformaldehyde in PBS for 10 min in the dark at room temperature.
4. Wash the fixed cells three times with PBS.
5. Add a minimal amount of fluorescence mounting medium on a microscope glass slide. Carefully transfer the coverslip from the 12-well plate onto the mounting medium with the sample facing the glass slide.
   NOTE: Seal the edge of the coverslip with nail polish if an aqueous mounting medium is used.

3. Measurement of Neurite Outgrowth

1. Use a 40x objective for capturing images using an epi-fluorescent microscope.
2. Capture images from at least 40 intact neurons with EGFP signal per transfection.
3. Open the captured image in ImageJ software with the NeuronJ plugin to measure the length of the longest neurite, from the cell body to the tip of the growth cone, of each imaged neuron.
4. Analyze the data obtained with the software to determine the effect of the targeted proteins in neurite outgrowth.