All lanes : Anti-Nestin antibody [2C1.3A11] (ab18102) at 1/5000 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : NES knockout HAP1 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 177 kDa

Lanes 1 - 2: Merged signal (red and green). Green - ab18102 observed at 300 kDa. Red - loading control, ab176560, observed at 50 kDa.

ab18102 was shown to recognize NES in wild-type HAP1 cells as signal was lost at the expected MW in NES knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and NES knockout samples were subjected to SDS-PAGE. Ab18102 and ab176560 (Rabbit anti-alpha Tubulin loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye^® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunofluorescence staining of U251 cells (glioblastoma cell line) stained using ab18102(green) and bisbenzimide (blue)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of migrating tumor cells labelling Nestin with ab18102 at 2µg/ml. Cytoplasm and membrane positive staining shown.

All lanes : Anti-Nestin antibody [2C1.3A11] (ab18102) at 1/1000 dilution

Lane 1 : Total rat brain protein at 5 µg
Lane 2 : Human CNS progenitor cell protein at 1 µg
Lane 3 : Human CNS progenitor cell protein at 5 µg

Predicted band size: 177 kDa

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of xenograft tumor cells labelling Nestin with ab18102.

Overlay histogram showing SH-SY5Y cells stained with ab18102 (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 (ab18102, 1/200 dilution) 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.

Immunohistochemistry of Anti-Nestin on formalin fixed paraffin embedded prostate nerve tissue using ab18102