This article details a protocol for immunofluorescence staining of neurons to visualize alpha-synuclein inclusions. The method involves several steps including fixation, permeabilization, and antibody incubation.
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 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.
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