Flow Cytometry - alpha Synuclein antibody [LB 509] (ab27766)

Overlay histogram showing PC12 cells stained with ab27766 (red line). The cells were fixed with methanol (5 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 (ab27766, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in PC12 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween used under the same conditions.

Western blot - alpha Synuclein antibody [LB 509] (ab27766)

Anti-alpha Synuclein antibody [LB 509] (ab27766) at 1 µg/ml + alpha Synuclein protein (ab51189) at 0.1 µg

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution

Performed under reducing conditions.

Predicted band size : 14 kDa
Observed band size : 15 kDa (why is the actual band size different from the predicted?)
Additional bands at : 13 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 20 minutes

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-alpha Synuclein antibody [LB 509] (ab27766)

IHC-P image of Alpha Synuclein (ab27766) staining in human brain samples with Multiple Systems Atrophy. 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 +18ºC to block non-specific protein-protein interactions. The sections were then incubated with ab27766 (1:400) for one hour at +18ºC, followed by Biotin conjugated anti-mouse goat secondary antibody (1/200). Formic acid pre-treatment (15min) revealed more inclusions in MSA tissue.

This image was taken from an abreview submitted by an anonymous reviewer.

Immunohistochemistry (PFA perfusion fixed frozen sections) - alpha Synuclein antibody [LB 509] (ab27766)

IHC-FoFr image of Alpha Synuclein staining on Synculein Transgenic mouse brain section stained using ab27766. The sections were incubated in 10% normal donkey serum in 0.1% PBS- and triton 0.3X100 for 1h to permeabilise the cells and block non-specific protein-protein interactions. The sections were then incubated with the antibody ab27766(1:500) overnight at +4ºC. Mouse secondary was Alexa 488 and Granular ferritin expression (Alex 568) seen in the cytosol.

Ruma Raha-Chowdhury, University Of Cambridge, United Kingdom

Western blot - Anti-alpha Synuclein antibody [LB 509] (ab27766)

Anti-alpha Synuclein antibody [LB 509] (ab27766) at 1/1000 dilution + alpha Synuclein protein (ab51189) at 0.1 µg

Secondary
Goat polyclonal Secondary Antibody to Mouse IgG - H&L (HRP), pre-adsorbed (ab97040) at 1/5000 dilution
developed using the ECL technique

Performed under reducing conditions.

Exposure time : 8 minutes