IHC image of alpha-synuclein staining in a section of formalin-fixed paraffin-embedded human Alzheimer's brain* performed on a Leica BOND™ system using the standard Protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab80627, 0.5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized ReNcell VM (Human neural progenitor) cells labeling Alpha-synuclein filament with ab209538 at 1/5000 dilution, followed by Alexa Fluor^® 647 Donkey anti-Rabbit IgG (H+L) secondary antibody at 1/400 dilution (red).

Upper panel counterstain: Anti-aggregated alpha-synuclein antibody clone 5G4 at 1/400 dilution, followed by AlexaFluor^®488 secondary detection (green).

Lower panel counterstain: Anti-alpha/beta-synculein antibody at 1/200 dilution, followed by Alexa Fluor^® 488 secondary detection (green).

Blocking buffer: 3% bovine serum albumin and 2% fetal bovine serum.

ReNcell VM cells were differentiated in media containing cAMP and GDNF (without bFGF or EGF) and transduced with Ad5C01 viral vector encoding human WT alpha-synuclein filament.

ab209538 detects alpha-synuclein filaments, which are also recognized by anti-aggregated alpha-synuclein antibody clone 5G4 (upper panel). 

Anti-alpha/beta-synculein antibody interacts with total alpha-synuclein in neurons, whereas ab209538 only recognizes the filamentous form (lower panel).    

Images were reproduced courtesy of Charles River.

IHC image of alpha Synuclein staining in normal Human cerebral cortex formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab138501, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

Human neuroblastoma cells stained for alpha-synuclein (green) using ab27766 in immunofluorescence. The neuroblastoma cells were fixed with paraformaldehyde and incubated with ab27766 (used at 5 μg/ml) for 12 hours at 4°C. A FITC conjugated Goat anti-Mouse IgG secondary antibody was then used.

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IHC-P image of Alpha Synuclein (ab27766) staining in human brain samples from patients with Multiple Systems Atrophy (MSA). The sections were subjected to heat-mediated antigen retrieval with citrate buffer. In addition, some slides received a 15 minute pre-treatment with Formic Acid. Sections were incubated in 20% serum for 30 minutes at +18°C to block non-specific protein-protein interactions. The sections were then incubated with ab27766 (1:400) for one hour at +18°C, followed by Biotin conjugated anti-mouse goat secondary antibody (1/200). Formic acid pre-treatment (15min) revealed more inclusions in MSA tissue.

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Immunocytochemistry/Immunofluorescence analysis of U87-MG (Human glioblastoma-astrocytoma epithelial cell line) cells labeling alpha Synuclein with purified ab138501 at 1/150 dilution (left panel). Cells were fixed with 4% paraformaldehyde. A Goat anti rabbit IgG(Alexa Fluor^®555) (1/200) was used as the secondary antibody. DAPI (blue) was used as the nuclear counter stain (right panel).

IHC image of alpha Synuclein (phospho S129) staining in Human Parkinson Substantia Nigra formalin fixed paraffin embedded tissue section*, performed on a Leica Bond™ system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab51253, 10µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset negative control image is taken from an identical assay performed on Human normal Substantia Nigra.