All lanes : Anti-Zic1 antibody [EPR7291(2)] (ab134951) at 1/2000 dilution (purified)

Lane 1 : Human brain lysates
Lane 2 : Human cerebellum lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 48 kDa

Blocking and diluting buffer: 5% NFDM/TBST. 

Flow Cytometry analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling Zic1 with purified ab134951 at 1:70 dilution (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor ® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Immunocytochemistry/ Immunofluorescence analysis of SH-SY5Y (Human neuroblastoma epithelial cell) cells labeling Zic1  with Purified ab134951 at 1:200 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. ab150077 Goat anti rabbit IgG(Alexa Fluor ® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Overlay histogram showing SH-SY5Y cells stained with unpurified ab134951 (red line). The cells were fixed with 80% 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 (ab134951, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 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 antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

All lanes : Anti-Zic1 antibody [EPR7291(2)] (ab134951) at 1/1000 dilution (unpurified)

Lane 1 : Human cerebellum tissue lysate
Lane 2 : Human fetal brain tissue lysate
Lane 3 : SH SY5Y cell lysate
Lane 4 : SW480 cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti rabbit at 1/2000 dilution

Predicted band size: 48 kDa

Immunofluorescent staining of SW480 cells labelling Zic1 with unpurified ab134951 at 1/100 dilution