Jun 03, 2026
In Situ α-Synuclein Seed Amplification Assay (iSAA) on Free-Floating Mouse Brain Sections
- Solji Choi1,
- Jayda Duvernay1,
- Bryan Killinger1
- 1Rush University
Protocol Citation: Solji Choi, Jayda Duvernay, Bryan Killinger 2026. In Situ α-Synuclein Seed Amplification Assay (iSAA) on Free-Floating Mouse Brain Sections. protocols.io https://dx.doi.org/10.17504/protocols.io.261gey7bov47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 14, 2026
Last Modified: June 03, 2026
Protocol Integer ID: 314954
Keywords: alpha-synuclein, preformed fibrils, animal model, seed amplification, synuclein seed amplification assay, floating mouse brain section, mouse brain sections this protocol, visualization of asyn species, asyn species, competent asyn species, detection of seed, floating tissue section, fibril
Funders Acknowledgements:
NIH
Abstract
This protocol describes the detection of seed-competent aSyn species in free-floating tissue sections from animal models treated with aSyn preformed fibrils (PFFs), enabling visualization of aSyn species associated with seeded aggregates. Following the assay, the signal localized to regions containing CIAP-resistant aggregated PS129.
Guidelines
Tissue collection for this protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee.
Materials
Critical Buffers
PIPES (200 mM, pH 6.9) Store at 4°C
- (200 mM)(0.2L)(302.37 g/mol) = 12.1 g PIPES
- PIPES will not readily dissolve until the solution is raised above pH 6.5. Slowly pH solution, if desired pH is passed then start over. DO NOT add acid to back pH.
Citrate Buffer (10mM Citric Acid, 0.05% Tween 20, pH 6.0) Store at 4°C
- 1.92 g Citric acid (anhydrous)
- 1000 mL Distilled water
- Mix to dissolve. Adjust pH to 6.0 with 1N NaOH and then add 0.5 ml of Tween 20 and mix well.
PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4)
TBS (150 mM NaCl, 50 mM, Tris-HCl, pH 7.6)
Day 1
18h 15m
Blocking endogenous peroxidase
Wash sections in TBS (1/2) 00:05:00
- TBS (pH 7.6)
| A | B | |
| NaCl | 150 mM | |
| Tris-HCl | 50 mM |
5m
Wash sections in TBS (2/2) 00:05:00
5m
Block sections in 0.3% hydrogen peroxide in TBS at RT. 00:30:00
30m
Heat-induced antigen retrieval
Heat citrate buffer to boiling in a microwave. Place the staining dish containing the sections into the hot citrate buffer.
Citrate buffer, pH 6.0
| A | B | |
| Citric Acid | 10mM | |
| Tween 20 | 0.05% |
Maintain the temperature by placing the container in a water bath set to 98°C 00:10:00
10m
Cool on ice until room temperature is reached.
15m
Buffer preconditioning
Wash sections in PBS (1/2) 00:05:00
PBS (pH 7.4)
| A | B | |
| NaCl | 137 mM | |
| KCl | 2.7 mM | |
| Na2HPO4 | 10 mM | |
| KH2PO4 | 1.8 mM |
5m
Wash sections in PBS (2/2) 00:05:00
5m
Add 100 mM PIPES buffer to each staining dish and incubate at RT01:00:00
*Dilute the 200 mM PIPES stock buffer to 100 mM before use.
PIPES buffer stock (200 mM, pH 6.9) Store at 4°C
***PIPES will not readily dissolve until the solution is raised above pH 6.5. Slowly pH solution, if desired pH is passed, then start over. DO NOT add acid to back pH.
1h
Monomer incubation (overnight on thermomixer)
Set the thermomixer (Eppendorf ThermoMixer F2.0) to 37°C.
Transfer each sample to a 1.5 mL LoBind Eppendorf tube.
Add recombinant His-tagged mouse αSyn monomer (ACRO Biosystems; Cat# ALN-M52H6) diluted in PIPES buffer, to a final concentration of 0.025 mg/mL. For the negative control, add PIPES buffer only.
Incubate samples overnight at 37°C on the thermomixer with the heated lid closed to prevent condensation on the tube caps.16:00:00
16h
Day 2
3h 44m
Fixation
Transfer samples from the 1.5 mL LoBind Eppendorf tube to a staining dish.
Wash sections in TBS (1/3) 00:03:00
3m
Wash sections in TBS (2/3) 00:03:00
3m
Wash sections in TBS (3/3) 00:03:00
3m
Incubate sections in 4% PFA at RT 00:10:00
*Use freshly prepared 4% PFA. Alternatively, prepare small aliquots, freeze them, and thaw one aliquot before each use.
10m
Washing and blocking
Wash tissues in PBS containing 0.5% Tween-20 (1/2)00:05:00
5m
Wash tissues in PBS containing 0.5% Tween-20 (2/2)00:05:00
5m
Block sections in 10% normal horse serum and 0.5% Triton X-100 in TBS. 01:00:00
1h
Primary antibody incubation
Dilute the HRP-conjugated anti-6×His antibody (Thermo Fisher Scientific, Cat# MA1-21315-HRP; 1:5,000) in the same blocking buffer and incubate at room temperature.02:00:00
2h
Detection
Wash tissues in PBS containing 0.5% Tween-20 (1/2)00:05:00
5m
Wash tissues in PBS containing 0.5% Tween-20 (2/2)00:05:00
5m
Wash tissues in pH-adjusted imidazole buffer for nickel-enhanced DAB detection (bright-field imaging) or in borate buffer for tyramide fluorophore detection (confocal fluorescence imaging).00:05:00
5m
Detect His-tagged aSyn immunoreactivity