Oct 30, 2024
Immunoperoxidase detection of human alpha-synuclein in mouse brain
- 1German Center for Neurodegenerative Diseases (DZNE)
Protocol Citation: Pietro La Vitola 2024. Immunoperoxidase detection of human alpha-synuclein in mouse brain . protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqd975vk5/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: October 30, 2024
Last Modified: October 30, 2024
Protocol Integer ID: 111266
Keywords: human alpha-synuclein
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000420
Abstract
This protocol is designed for detecting human alpha-synuclein (Abcam, RRID:AB_2537217, MJFR1). Tissues stained with this protocol include 35 μm free-floating mouse brain sections. All tissues were from mice perfused with 4% PFA.
Immunoperoxidase detection of human alpha-synuclein in mouse brain samples
Immunoperoxidase detection of human alpha-synuclein in mouse brain samples
Wash tissue slices in tris-buffer saline (TBS: 0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes. Repeat x3.
Incubate in in 10% Methanol + 3% H2O2 in TBS for 20 min at room temperature
Wash tissue slices in tris-buffer saline (TBS: 0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes. Repeat x3.
Incubate in blocking buffer (5% goat serum, 2% BSA, 0.5% triton X-100 in TBS) for 1 hour at room temperature.
Incubate with anti-human-alpha-synuclein primary antibody (RRID:AB_2537217) at 1:5000 dilution in 50% blocking solution for 24 hours at room temperature.
Wash tissue slices in TBS-T (TBS+ 0.25% Triton x-100) for 10 minutes. Repeat x3.
Incubate in goat anti-rabbit biotinylated secondary antibody at 1:250 dilution in 50% blocking buffer for 1 hour at room temperature.
Wash tissue slices in TBS-T(TBS+ 0.25% Triton X-100) for 10 minutes. Repeat x 2.
Wash tissue slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes.
Incubate 1 hour with ABC complex. ABC complex to be prepared 30 min prior to use.
Wash tissue slices in TBS-T(TBS+ 0.25% Triton X-100) for 10 minutes. Repeat x 2.
Wash tissue slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes.
Stain with the peroxidase chormogen diaminobenzidine
Wash tissue slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes. Repeat X 3.