Immunostaining of Neurons Treated with Alpha-Synuclein Aggregates

Begin with an adherent neuron culture treated with alpha-synuclein or alpha-S aggregates, which are clumps of a misfolded protein.

These clumps aggregate to form intracellular inclusions.

Replace the media with a fixing solution to preserve cellular integrity.

Remove the fixing solution and wash with buffer.

Next, add a surfactant to permeabilize the cellular membrane.

Remove the surfactant and wash with buffer.

Introduce a blocking solution to prevent non-specific antibody binding.

Now, incubate with primary antibodies that bind specifically to alpha-S inclusions and structural proteins.

Wash with buffer to remove unbound antibodies.

Incubate with fluorescently labeled secondary antibodies.

Remove unbound antibodies.

Add a fluorescent stain to label the nucleus.

Remove the stain and wash with buffer.

Using mounting media, mount the coverslip with neurons on a slide.

Under a fluorescent microscope, observe immunolabeled alpha-S inclusions within the neuron.

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