Recombinant Anti-Alpha-synuclein aggregate antibody [MJFR-14-6-4-2] - Conformation-Specific (ab209538)

Clone MJFR-14-6-4-2 (ab214033) has been successfully conjugated by Abcam. This image was generated using Anti-Alpha-synuclein aggregate antibody [MJFR-14-6-4-2] - Conformation-Specific (Alexa Fluor® 488). Please refer to ab216124 for protocol details.

IHC image of alpha-synuclein aggregate staining in a section of frozen Parkinson human substantia nigra*.

The section was fixed using 10% formaldehyde in 1XPBS for 10 minutes. No antigen retrieval step was performed prior to staining. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature.  The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab216124 at 1/100 dilution (shown in green) and counterstained using ab195884, Rat monoclonal to Tubulin (Alexa Fluor^® 647), at 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

Clone MJFR-14-6-4-2 (ab214033) has been successfully conjugated by Abcam. This image was generated using Anti-Alpha-synuclein aggregate antibody [MJFR-14-6-4-2] - Conformation-Specific (Alexa Fluor® 647). Please refer to ab216309 for protocol details.

IHC image of alpha-synuclein aggregate staining in a section of formalin-fixed paraffin-embedded Parkinson human substantia nigra*.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Biocare Medical NxGen pressure cooker using retrieval settings of 110^oC for 8 minutes. Non-specific protein-protein interactions were then blocked in TBS containing 0.025% (v/v) Triton X-100, 0.3M (w/v) glycine and 1% (w/v) BSA for 1h at room temperature.  The section was then incubated overnight at +4°C in TBS containing 0.025% (v/v) Triton X-100 and 1% (w/v) BSA with ab216309 at 1/5000 dilution (shown in red) and counterstained using ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue). The section was then mounted using Fluoromount^®.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

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

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

ab209538 staining Alpha-synuclein aggregate in rat dorsal root ganglian (DRG) tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde, cut into 20 micron slices and blocked with 2% BSA for 10 minutes at 21°C and antigen retrieval was by heat mediation in citrate buffer. The sample was incubated with primary antibody (1/2000 in TBS/BSA/azide) at 21°C for 2 hours. An Alexa Fluor® 555-conjugated Goat anti-rabbit polyclonal (1/300) was used as the secondary antibody.

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Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.2% Triton X-100 permeabilized ReNcell VM (Human neural progenitor) cells labeling Alpha-synuclein aggregate 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.

Images were reproduced courtesy of Charles River.

ab209538 staining Alpha-synuclein aggregate in mouse colon tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde, cut into 20 micron slices, blocked with 2% BSA for 10 minutes at 21°C and antigen retrieval was by heat mediation in citrate buffer. The sample was incubated with primary antibody (1/2000 in TBS/BSA/azide) at 21°C for 2 hours. A biotin-conjugated goat anti-rabbit polyclonal (1/300) was used as the secondary antibody.

See Abreview

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

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 alpha-synuclein filament (Left image). Right image show control vector cells.

Images were reproduced courtesy of Charles River.

Ab209538 staining Alpha-synuclein aggregate in Human DLB brain tissue sections by Immunohistochemistry (Formalin/PFA fixed paraffin embedded sections). Tissue was fixed with paraformaldehyde and blocked with 2% BSA for 10 minutes at 21°C; antigen retrieval was by heat mediation in Citric acid. Samples were incubated with primary antibody (1/10,000 in TBS) for 2 hours at 21°C. A biotin conjugated anti-rabbit IgG Goat polyclonal was used as the secondary antibody at 1/300 dilution.

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Dot blot analysis of alpha-synuclein filament labeled with ab209538 at 2.2 ng/ml.

Lane 1: Recombinant alpha-synuclein filament treated with 70% formic acid.

Lane 2: Recombinant alpha-synuclein filament treated with 70% formic acid.

Lane 3: Untreated recombinant alpha-synuclein filament.

Lane 4: Untreated recombinant alpha-synuclein filament.

Alpha Synuclein filaments were generated using full length recombinant alpha-synuclein (aa1-140) by incubation in 20mM TRIS, pH 7.2 at 37 °C with agitation.

Denaturation of filaments with 70% formic acid reduces antibody recognition by 30-100 fold, demonstrating conformation specificity.

Data is provided by Professor Poul Henning Jensen, Aarhus University, Denmark.

Dot Blot showing the reactivity of ab209538 (2 ng/ml) with 

F: alpha synuclein Filament

F+FA: alpha synuclein Filament treated in 50% formic acid for 1 h 37oC prior to application to the dot blot.

M+FA: alpha synuclein Monomer treated in 50% formic acid for 1 h 37oC prior to application to the dot blot.

Loading control antibody (1:1000) reacts with Alpha-synuclein irrespectively of it being in a filament, oligomer or a monomer.