Feb 22, 2024
Protocol for "immunoperoxidase detection of alpha-synuclein in non-human primate brain samples
- 1Center for Applied Medical Research (Cima) University of Navarra
Protocol Citation: jlanciego 2024. Protocol for "immunoperoxidase detection of alpha-synuclein in non-human primate brain samples. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5x4wdg1b/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: February 22, 2024
Last Modified: February 22, 2024
Protocol Integer ID: 95605
Keywords: Immunoperoxidase, diaminobenzidine, ABC method, immunohistochemistry, brain
Funders Acknowledgement:
Aligning Science Across Parkinson's through the Michael J. Fox Foundation for Parkinson's Research
Grant ID: ASAP-020505
Disclaimer
Noting to be disclosed
Abstract
Here an step-by-step protocol is provided summarizing the immunoperoxidase detection of neurons expressing alpha-synuclein in the macaque brain. Enclosed image illustrates layer V pyramidal neurons showing immunoreactivity for alpha-synuclein at the level of the cerebral cortex (insular gyrus).
Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
Frozen coronal sections (40 microns-thick) to be obtained in a sliding microtome (Leica HM400) and collected in 0.125 M phosphate buffer (PB) pH 7.4 as 10 series of adjacent sections.
Wash the sections three times (10 min each) in phosphate-buffered saline solution (PBS; 0.05M pH 7.4)
Inactivate the endogenous peroxidase activity: incubation for 40 min in a solution made of 10 ml ethanol, 600 microliters of H2O and 133 microliters of H2O2.
Preincubation solution (60 min): 1% bovine serum albumin (BSA) + 0.05% triton X-100 + 0.05% gelatin.
Incubation with the primary antibody (overnight at 4 ºC). Mouse anti-alpha-synuclein (monoclonal), 1:40 in PBS.
Wash the sections three times (10 min each) in PBS 0.05M pH 7.4
Incubation with the secondary antibody (120 min). Donkey anti.mouse (Biotin-SP), 1:600 in PBS.
Wahs the sections three times (10 min each) in PBS 0.05M pH 7.4
Incubation with ABC complex (90 min). ABC complex to be prepared 30 min prior to use.
Wash the sections three times (10 min each) in PBS 0.05M pH 7.4
Stain with the peroxidase chormogen diaminobenzidine (DAB; 3-5 min). Incubation solution made of 0.05% DAB and 0.01% H2O2 in PBS 0.05M pH 7.4
Wash the sections three times 810 min each) in PBS 0.05M pH 7.4
Mount free-floating sections in gelatin-coated slides and air-dry overnight.
Sections to be dehydrated and cleared in 100% toluene (2 x 10 min) and coverslipped with DePex.