TITLE: Aggregated aSyn Dot Blot assay
AUTHORS: rabdelha  

[DESCRIPTION]
This protocol details aggregated aSyn Dot Blot assay.

[STEPS]
SECTION: Aggregated aSyn Dot Blot assay
1. Normalize protein samples to equal concentrations between 100 ng-1000 ng/μl in PBS.

SECTION: Aggregated aSyn Dot Blot assay
2. Carefully spot 1.5 µL each sample onto dry nitrocellulose (  pores) membrane.

SECTION: Aggregated aSyn Dot Blot assay
3. Allow moisture to dry for a minute or two.
SECTION: Aggregated aSyn Dot Blot assay
4. Block membrane in 5% skim milk in TBST.
SECTION: Aggregated aSyn Dot Blot assay
5. Wash membrane 1X in TBST.
SECTION: Aggregated aSyn Dot Blot assay
6. 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).
SECTION: Aggregated aSyn Dot Blot assay
7. Incubate at Room temperature for 1-2 hours, or 60 min at 4 °C.

SECTION: Aggregated aSyn Dot Blot assay
8. Wash 3x with TBST.
SECTION: Aggregated aSyn Dot Blot assay
9. Incubate with anti-rabbit IgG (or appropriate secondary)at 1:1000 for 1-2 hours in TBST.
SECTION: Aggregated aSyn Dot Blot assay
10. Wash 3x with TBST.
SECTION: Aggregated aSyn Dot Blot assay
11. Develop with Biorad Chemiluminescent substrate, or similar.
SECTION: Proteinase K resistant aSyn histology
12.  
 

Wash 50um brain sections 1x in PBS.
SECTION: Proteinase K resistant aSyn histology
13. Treat with Protinase K (NEB, 1:4000, stock at 20 mg/mL) for 10 min at Room temperature.

SECTION: Proteinase K resistant aSyn histology
14. Wash 2x with IHC buffer (3% NGS, 0.3% TX100, in PBS).
SECTION: Proteinase K resistant aSyn histology
15. Block with Mouse-on-Mouse kit (Vector Lab, PK-2200, just first step in instructions) for 60 min.

SECTION: Proteinase K resistant aSyn histology
16. Wash 2x with IHC buffer.
SECTION: Proteinase K resistant aSyn histology
17. Incubate   at 4 °C with primary antibody 1:250 – 1:1000 (anti-synuclein BD Biosci 610787).

SECTION: Proteinase K resistant aSyn histology
18. Wash 3x with PBS.
SECTION: Proteinase K resistant aSyn histology
19. Incubate with desired secondary (anti-Mouse IgG AF488 is my choice at 1:1000).
SECTION: Proteinase K resistant aSyn histology
20. Wash 3x with PBS.
SECTION: Proteinase K resistant aSyn histology
21. Stain with Nissl and Mount with Profade with DAPI.
SECTION: Proteinase K resistant aSyn histology
20.1. Wash with PBS (1/3).
SECTION: Proteinase K resistant aSyn histology
20.2. Wash with PBS (2/3).
SECTION: Proteinase K resistant aSyn histology
20.3. Wash with PBS (3/3).
SECTION: Proteinase K resistant aSyn histology
18.1. Wash with PBS (1/3).
SECTION: Proteinase K resistant aSyn histology
18.2. Wash with PBS (2/3).
SECTION: Proteinase K resistant aSyn histology
18.3. Wash with PBS (3/3).
SECTION: Proteinase K resistant aSyn histology
16.1. Wash with IHC buffer (1/2).
SECTION: Proteinase K resistant aSyn histology
16.2. Wash with IHC buffer (2/2).
SECTION: Proteinase K resistant aSyn histology
14.1. Wash with IHC buffer (3% NGS, 0.3% TX100, in PBS) (1/2).
SECTION: Proteinase K resistant aSyn histology
14.2. Wash with IHC buffer (3% NGS, 0.3% TX100, in PBS) (2/2).
SECTION: Aggregated aSyn Dot Blot assay
10.1. Wash with TBST (1/3).
SECTION: Aggregated aSyn Dot Blot assay
10.2. Wash with TBST (2/3).
SECTION: Aggregated aSyn Dot Blot assay
10.3. Wash with TBST (3/3).
SECTION: Aggregated aSyn Dot Blot assay
8.1. Wash with TBST (1/3).
SECTION: Aggregated aSyn Dot Blot assay
8.2. Wash with TBST (2/3).
SECTION: Aggregated aSyn Dot Blot assay
8.3. Wash with TBST (3/3).