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 fibrils to each experimental well, and 3.75 microliters of PBS to each control well.

When working with PFFs, be cautious about the unwanted protein contamination and afterwards, clean the hood and all PFF-associated instruments with 1% SDS and 70% ethanol.

At the appropriate experimental endpoint, after staining for the markers of interest, load the 96 well culture plate onto a high content plate scanner, fitted with a 10x objective. Adjust the settings according to the specifications of the 96 well plate, such as the plate type, manufacturer, size, distance between the wells, and the type and volume of medium.

Select the imaging area of the wells to cover all of the cells in each micro island, and use one well to adjust the autofocus on the DAPI expression. Calibrate the acquisition time for each fluorescent channel, based on the intensity of the staining in the control wells, and adjust the parameters so that the dopamine cells in the preformed fibril treated control wells, harboring phosphoserine 129 alpha synuclein aggregates within the cell soma, clearly be distinguished, allowing an unambiguous quantification of the phosphoserine alpha synuclein positive and negative cells.

Then, image all of the selected wells simultaneously in all of the channels, using exactly the same parameters for each well.