Immunostaining of Neurons Treated with Alpha-Synuclein Aggregates

To perform immunofluorescence in a chemical fume hood fix the neurons with 2% paraformaldehyde in PBS and 5% sucrose solution for 15 minutes at room temperature without shaking. After 15 minutes, remove the fixing solution, and briefly wash the neurons with PBS three times.

Next, permeabilize the neurons with 0.3% of nonionic surfactant in PBS for 5 minutes at room temperature. Then, briefly wash the neurons with PBS three times. Incubate the neurons with 3% FBS in PBS for 30 minutes at room temperature on an orbital shaker to block unspecific binding sites. Afterward, incubate the neurons with appropriate primary antibody dissolved in 3% FBS in PBS overnight in the cold room or refrigerator overnight at 4 degrees Celsius on an orbital shaker.

The next day, remove the antibody solution and wash the cells briefly with PBS for three times. Then, incubate the neurons with the appropriate fluorescent secondary antibody dissolved in 3% FBS in PBS for 1 hour at room temperature in the dark on an orbital shaker.

Remove the antibody solution and wash briefly with PBS for three times. Next, stain the neurons with DAPI solution for 15 minutes at room temperature in the dark on an orbital shaker. After 15 minutes, remove the antibody solution and wash briefly with PBS for three times. Mount the coverslips on the slide using anti-fade mounting medium.