The following protocol was used to generate the ICC images of neurons treated with type 1 and type 2 alpha-synuclein preformed fibrils (PFFs).  Primary rat hippocampal neurons show lewy body inclusion formation when treated with type 1 alpha-synuclein PFFs but not when treated with type 2 alpha-synuclein PFFs.

1. Hippocampal neurons harvested from postnatal day 1 Sprague Dawley rat pups and plated at 80,000 cells per well on a poly-D-lysine and laminin-coated plate.
2. Preformed fibrils were sonicated for 1 hour in a water-bath sonicator immediately prior to treatment.
3. Neurons were treated with preformed fibrils at 4 µg/mL. Control wells with vehicles were maintained.
4. All wells underwent a 50% media change after 7 days.
5. All wells were fixed with 4% formaldehyde for 20 minutes 7 days after the media change (day-in-vitro 14 after treatment).
6. The formaldehyde was washed off with three washes of filtered 10 mM phosphate-buffered saline (10 minutes each).
7. Cells were then blocked with 1:1 PBS: LiCOR Odyssey Block (LiCOR, 927-40010) and 30 uL/mL of 0.1% triton-X 100 for 30 mins.
8. After 30 mins, primary antibodies diluted in 1:1 PBS: LiCOR Odyssey Block (with 30 uL/mL of 0.1% triton-X 100) were added to the wells and incubated overnight at 4 degrees.
9. The next day, the primary antibody solution was washed off with three washes of filtered 10 mM phosphate-buffered saline (10 minutes each).
10. The secondary antibody was diluted in 1:1 PBS: LiCOR Odyssey Block (with 30 uL/mL of 0.1% triton-X 100) and added to the wells for 60 mins in the dark.
11. After 60 mins, the secondary antibody solution was washed off with three washes of filtered 10 mM phosphate-buffered saline (10 minutes each).
12. The plate was then imaged on an epifluorescent microscope (Olympus IX73, B&B Microscopes) at 20X objective.
13. All images were captured at the same fixed scaling and same exposure times.

 

Primary antibody: Anti-pSer129

Counterstain: Hoechst reagent

Counterstain dilution: 1:3000

Counterstain incubation time: 1 hr