Studying Alpha-Synuclein Accumulation in Primary Embryonic Mouse Dopamine Neurons

Take micro-island cultures of primary embryonic mouse dopamine neurons in a multi-well plate containing media.

These neurons are pre-treated with pre-formed fibrils (PFFs) — misfolded and aggregated forms of the alpha-synuclein protein.

PFFs act as seeds, triggering further misfolding and oligomerization of endogenous alpha-synuclein, forming phosphorylated alpha-synuclein fibrils. These fibrils eventually form Lewy bodies.

Replace the media with a fixative to preserve cellular morphology.

Wash the cells with buffer.

Introduce a detergent-containing buffer to permeabilize the cell membranes.

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

Add primary antibodies targeting phosphorylated alpha-synuclein and a reference protein ubiquitously expressed in dopamine neurons.

Wash to remove unbound antibodies.

Introduce fluorophore-conjugated secondary antibodies targeting the respective primary antibodies.

Wash again to remove unbound antibodies.

Add a DNA-binding dye to stain the nuclei.

Using a fluorescence plate scanner, quantify the dopamine neurons containing cytoplasmic alpha-synuclein aggregates.

Add a 3.75 microliters of 100 microgram per milliliter of pre-formed