Anti-alpha + beta Synuclein antibody [EP1646Y] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-alpha + beta Synuclein antibody [EP1646Y] - BSA and Azide free (AB189217)

Overlay histogram showing SH-SY5Y cells stained with unpurified ab51252 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab51252, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluorr^® 488 goat anti-rabbit IgG (H+L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51252).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-alpha + beta Synuclein antibody [EP1646Y] - BSA and Azide free (AB189217)

Overlay histogram showing U87-MG cells fixed in 4% PFA and stained with purified ab51252 at a dilution of 1 in 30 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line). This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab51252).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha + beta Synuclein antibody [EP1646Y] - BSA and Azide free (AB189217)

Clone EP1646Y (ab189217) has been successfully conjugated by Abcam. This image was generated using Anti-alpha + beta Synuclein antibody [EP1646Y] (Alexa Fluor® 647). Please refer to ab199304 for protocol details.

IHC image of alpha + beta Synuclein staining in a section of formalin-fixed paraffin-embedded normal human cerebellum.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6) in a Dako Pascal pressure cooker using the standard factory-set regime. 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 ab199304 at 1/50 (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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-alpha + beta Synuclein antibody [EP1646Y] - BSA and Azide free (AB189217)

Clone EP1646Y (ab189217) has been successfully conjugated by Abcam. This image was generated using Anti-alpha + beta Synuclein antibody [EP1646Y] (Alexa Fluor® 488). Please refer to ab199086 for protocol details.

ab199086 staining alpha+beta Synuclein in PC12 cells. The cells were fixed with 100% methanol (5min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab199086 at 1/50 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 594), at 2µg/ml (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

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

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-alpha + beta Synuclein antibody [EP1646Y] - BSA and Azide free (AB189217)

This ICC/IF data was generated using the same anti-alpha+beta synuclein antibody clone, EP1646Y, in a different buffer formulation (cat# ab51252).

Immunocytochemistry/Immunofluorescence analysis of PC-12 cells labelling alpha + beta Synuclein with purified ab51252 at 1/100. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).