Sep 09, 2024
human alpha-synuclein and aggregated alpha-synuclein immunofluorescence staining
- 1German Center for Neurodegenerative Diseases (DZNE)
Protocol Citation: Pietro La Vitola 2024. human alpha-synuclein and aggregated alpha-synuclein immunofluorescence staining. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov19om7lr2/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: May 17, 2024
Last Modified: September 09, 2024
Protocol Integer ID: 100021
Keywords: human alpha-synuclein, mouse brain slices, aggregated alpha-synuclein
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol is designed for human alpha-synuclein staining using the MJFR1-Alexa 488 (RRID:AB_2537217) antibody and either anti-SynO2 (RRID:AB_2632701) or MJFR-14-6-4-2 (RRID:AB_2714215) antibodies. Tissue stained with this protocol include 35 μm free-floating mouse brain sections. All tissue was from mice perfused with 4% PFA.
Day 1
Day 1
10m
10m
Rinse brain slices (35µ) in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) 3X 00:10:00
30m
Quench brain slices in a solution containing 3%H2O2 and 10% Methanol in TBS 00:20:00
20m
Rinse brain slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) 3X 00:10:00
30m
Incubate in blocking buffer (5% donkey serum, 2% BSA, 0.5% triton X-100 in TBS) 01:00:00 hour at room temperature
1h
Incubate in mouse on mouse blocking buffer (MKB-2213; Vector Lab) 00:20:00 at room temperature
20m
Rinse brain slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6)00:00:10
10s
Incubate with anti-SynO2 (1:1000; RRID:AB_2632701) or anti-MJFR-14-6-4-2 (1:20000;
RRID:AB_2714215) dilution in 50% blocking solution 48:00:00 hour at 4°C
2d
Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100)) 3X 00:10:00
30m
Incubate in fluorescent secondary antibody at 1:200 dilution in 50% blocking buffer 01:00:00 r at room temperature.
1h
Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100) 3X 00:10:00
30m
Incubate with rabbit anti-hASYN(1:400; RRID:AB_2537217, MJFR1-Alexa 488) 24:00:00 at 4°C in 50% blocking buffer
1d
Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100) 2x 00:10:00
20m
Rinse brain slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6)00:10:00
10m
Mount free-floating sections on SuperFrost+ slides (if staining free-floating tissue) and let dry at room temperature for 15 minutes.
Coverslip with fluorescent mounting medium and #1.5 coverslips. Outline coverslip with clear nail polish and store at 4oC .