Analysis of _-synuclein expression in formalin-fixed paraffin-embedded (FFPE) human Substantia Nigra tissue by Immunohistochemistry (IHC) using sheep antibody to _-synuclein (S-075-50, 4 µg/mL). Sections were cut to 4 um thickness and antigen retrieval performed using citrate buffer, pH 6, for 20 minutes. Primary antibody binding was visualized with a donkey anti-sheep-HRP conjugated secondary antibody (1:100) and 3'3-diaminobenzidine (DAB, brown). Cell nuclei were stained with a Hematoxylin nuclear counterstain (blue).
Immunohistochemical staining of lewy body in Parkinsons diseased human brain tissue using sheep polyclonal to human alpha synuclein, catalogue number S-075-50.
Analysis of alpha-synuclein expression by Flow Cytometry (A), Immunocytochemistry (B) and Western Blotting (C). The Flow Cytometry histogram (A) demonstrates presence of alpha-synuclein (blue line) in human SHSY-5Y neuroblastoma cells. Specific alpha-synuclein immunoreactivity (IR) is also observed in SHSY-5Y cells by Immunocytochemistry (B1, green), while under control conditions (B2), alpha-synuclein-IR is absent. In rodent tissue, the antibody detects _-synuclein monomer (~15 kDa) and a band at ~50 kDa on the blot, which most likely represents multimeric _-synuclein protein, which has been described by Shen et al., 2015, and others. Both bands are _-synuclein antibody specific as demonstrated by blocking with immunizing peptide. Recombinant human _-synuclein protein (100 ng) was run as control. Flow Cytometry method: Fixing and permeabilization: Absolute methanol (10 minutes on ice) and 0.1% Tween-20 in PBS; Blocking: 200 µg/mL sheep IgG; Primary antibody: sheep antibody to alpha-synuclein (S-075-50, 2 µg per ~10^6 cells, blue) for 30 minutes at room temperature; Secondary antibody: Goat anti-sheep, PE-labeled (1:100 dilution), 20 minutes in dark at room temperature. Negative control: Normal sheep IgG (S-1754-500, black). Data and results were generated using Orflo MoxiflowTM instrument and protocols. Immunocytochemistry method: Fixed (4% formaldehyde), permeabilized, and blocked (10% normal horse serum, 0.1% Triton X100) cells were incubated with alpha-synuclein antibody S-075-50 (2 µg/mL, green) for 1 hour. Primary antibody binding was visualized with a secondary donkey anti-sheep-CF488A antibody (4 µg/mL, 1 hour incubation). Cell nuclei were stained with Hoechst dye (blue). Control cells were stained following the same protocol, substituting the primary antibodies with normal sheep IgG (S-1754-500). Magnification: 100x. Western Blotting method: Protein loading amount: 50 µg per lane; Transfer: Towbin's buffer with 20% methanol, semi-dry; Blocking: 5% skim milk; Primary antibody: 1 µg/mL, overnight incubation at 2-8°C; Secondary antibody: anti-sheep-HRP (1:6000); Detection: Chemiluminiscence. Proteins were fixed on membrane (0.4% formaldehyde, 30 minutes at room temperature) after transfer as described by Lee & Kamitani, 2011.