Sep 09, 2024

Public workspaceSOD2 and human alpha-synuclein immunofluorescence staining

 Forked from  human alpha-synuclein and aggregated alpha-synuclein immunofluorescence staining
DOI
dx.doi.org/10.17504/protocols.io.6qpvr8zxblmk/v1
  • 1German Center for Neurodegenerative Diseases (DZNE)
Pietro La Vitola
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DOI: dx.doi.org/10.17504/protocols.io.6qpvr8zxblmk/v1
Protocol Citation: Pietro La Vitola 2024. SOD2 and human alpha-synuclein immunofluorescence staining. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8zxblmk/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: September 09, 2024
Last Modified: September 09, 2024
Protocol Integer ID: 107158
Keywords: human alpha-synuclein, mouse brain slices, SOD2
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol is designed for SOD2 and human alpha-synuclein staining using the anti-SOD2 (RRID:AB_2636921) and MJFR1-Alexa 488 (RRID:AB_2537217) 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 Duration00:10:00

30m
Quench brain slices in a solution containing 3%H2O2 and 10% Methanol in TBS Duration00:20:00
20m
Rinse brain slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) 3X Duration00:10:00

30m
Incubate in blocking buffer (5% donkey serum, 2% BSA, 0.5% Triton X-100 in TBS) Duration01:00:00 hour at room temperature
1h
Incubate with anti-SOD2 (1:1000; Cell Signaling, RRID:AB_2636921) in 50% blocking solution Duration24:00:00 hour at 4°C
1d
Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100) 3X Duration00:10:00
30m
Incubate in fluorescent secondary antibody at 1:200 dilution in 50% blocking buffer Duration01:00:00 r at room temperature.
1h
Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100) 3X Duration00:10:00
30m
Incubate with rabbit anti-hASYN(1:400; RRID:AB_2537217, MJFR1-Alexa 488) Duration24:00:00 at 4°C in 50% blocking buffer
1d
Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100) 2x Duration00:10:00
20m
Rinse brain slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6)Duration00:10:00
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 .