TITLE: Protocol for &#34;immunoperoxidase detection of alpha-synuclein in non-human primate brain samples
AUTHORS: jlanciego

[DESCRIPTION]
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).

[STEPS]
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
1. 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.
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
2. Wash the sections three times (10 min each) in phosphate-buffered saline solution (PBS; 0.05M pH 7.4)
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
3. 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.
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
4. Preincubation solution (60 min): 1% bovine serum albumin (BSA) + 0.05% triton X-100 + 0.05% gelatin.
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
5. Incubation with the primary antibody (overnight at 4 ºC). Mouse anti-alpha-synuclein (monoclonal), 1:40 in PBS.
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
6. Wash the sections three times (10 min each) in PBS 0.05M pH 7.4
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
7. Incubation with the secondary antibody (120 min). Donkey anti.mouse (Biotin-SP), 1:600 in PBS.
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
8. Wahs the sections three times (10 min each) in PBS 0.05M pH 7.4
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
9. Incubation with ABC complex (90 min). ABC complex to be prepared 30 min prior to use.
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
10. Wash the sections three times (10 min each) in PBS 0.05M pH 7.4
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
11. 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
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
12. Wash the sections three times 810 min each) in PBS 0.05M pH 7.4
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
13. Mount free-floating sections in gelatin-coated slides and air-dry overnight.
SECTION: Immunoperoxidase protocol (all steps below are conducted in free-floating sections and at room temperature unless otherwise noted)
14. Sections to be dehydrated and cleared in 100% toluene (2 x 10 min) and coverslipped with DePex.