All lanes : Anti-Lamin A antibody [133A2] (ab8980) at 1/100 dilution

Lane 1 : Wild-type HAP1 whole cell lysate
Lane 2 : LMNA knockout HAP1 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : HepG2 whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 74 kDa
Observed band size: 74 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab8980 observed at 74 kDa. Red - loading control, ab181602, observed at 37 kDa.

ab8980 was shown to recognize  in wild-type HAP1 cells as signal was lost at the expected MW in LMNA knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and LMNA knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3pc Milk. Ab8980 and ab181602 (Rabbit anti GAPDH loading control) were incubated overnight at 4°C at 1/100 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.

ICC/IF image of ab8980 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum (ab7481) / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab8980 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

ab8980 staining of Lamin A in adult mouse liver sections. Formaldehyde-fixed paraffin-embedded sections of mouse liver tissue were incubated with ab8980 (1/2500) fo 2 hours. Antigen retrieval was performed by heat induction in citrate buffer pH 6.0 (ab64214). Please see accompanying abreview for additional information.

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Overlay histogram showing HeLa cells stained with ab8980 (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 (ab8980, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG3 [MG3-35] (2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a decreased signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween used under the same conditions.

These images show HeLa cells stained with Laminin A antibody - ab8980 (green) and with DAPI (blue). The pictures were kindly supplied as part of the review submitted by Dr Josef Gotzmann at Medical University of Vienna, Austria.

Immunohistochemistry on paraffin section of human colon.

Immunohistochemistry on frozen sections of human colon showing nuclear lamina staining in epithelial and connective tissue cells.

Immunocytochemical staining of fiboblasts showing nuclear lamina