Aggregated aSyn Dot Blot assay

Normalize protein samples to equal concentrations between 100ng-1000 in PBS.

Carefully spot 1.5µL each sample onto dry nitrocellulose (0.45 µm pores) membrane.

Allow moisture to dry for a minute or two.

Block membrane in 5% skim milk in TBST.

Wash membrane 1X in TBST.

Apply anti-aggregated aSyn antibody in 5% skim milk in TBST (1:1000 for Abcam fibril.aggregate specific aSyn antibody: MJFR 14-6-4-2).

Incubate at Room temperature for 1-2 hours, or 1h 0m 0s at 4°C.

Wash 3x with TBST.

Wash with TBST (1/3).

Wash with TBST (2/3).

Wash with TBST (3/3).

Incubate with anti-rabbit IgG (or appropriate secondary)at 1:1000 for 1-2 hours in TBST.

Wash 3x with TBST.

Wash with TBST (1/3).

Wash with TBST (2/3).

Wash with TBST (3/3).

Develop with Biorad Chemiluminescent substrate, or similar.

Wash 50um brain sections 1x in PBS.

Treat with Protinase K (NEB, 1:4000, stock at 20mg/mL) for 0h 10m 0s at Room temperature.

Wash 2x with IHC buffer (3% NGS, 0.3% TX100, in PBS).

Wash with IHC buffer (3% NGS, 0.3% TX100, in PBS) (1/2).

Wash with IHC buffer (3% NGS, 0.3% TX100, in PBS) (2/2).

Block with Mouse-on-Mouse kit (Vector Lab, PK-2200, just first step in instructions) for 1h 0m 0s.

Wash 2x with IHC buffer.

Wash with IHC buffer (1/2).

Wash with IHC buffer (2/2).

Incubate at 4°C with primary antibody 1:250 – 1:1000 (anti-synuclein BD Biosci 610787).

Wash 3x with PBS.

Wash with PBS (1/3).

Wash with PBS (2/3).

Wash with PBS (3/3).

Incubate with desired secondary (anti-Mouse IgG AF488 is my choice at 1:1000).

Wash 3x with PBS.

Wash with PBS (1/3).

Wash with PBS (2/3).

Wash with PBS (3/3).

Stain with Nissl and Mount with Profade with DAPI.