Immunocytochemical analysis of U2OS cells, labeling Nup153 with ab24700. Cells were formaldehyde fixed, permeabilized with NP40, and blocked with 3% BSA for 1 hour at 21°C. Immunostaining with ab24700 diluted 1/100 for 12 hours at 4°C.

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Human and mouse cells treated with different concentrations of ab24700. The cells were fixed in 4% formaldehyde and permeabilized in 0.2% Triton x100 for 10 minutes at room temperature, then washed 3 times in PBS. The fixation and permeabilization was carried out in a single step. The image shows clear staining of the nuclear envalope (green). The DNA is stained with DAPI (blue).

A: HeLa cells, ab24700 used at 4µg/ml
B: 3T3 cells, ab24700 used at 2µg/ml
C: 3T3 cells, ab24700 used at 4µg/ml
D: 3T3 cells, ab24700 used at 6.6µg/ml

Overlay histogram showing HeLa cells stained with ab24700 (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 (ab24700, 2µg/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.

ICC/IF image of ab24700 stained HepG2 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 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 (ab24700, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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.

Methanol fixed HeLa stained with ab24700. This antibody brilliantly highlights the nuclear membrane (green). The golgi is stained with Giantin (yellow).

Human and mouse cells treated with different concentrations of ab24700. The cells were fixed in 100% methanol for 20 minutes at -20°C, then washed once in PBS. The image shows clear staining of the nuclear envalope (green). The DNA is stained with DAPI (blue).

A: HeLa cells, ab24700 used at 4µg/ml
B: 3T3 cells, ab24700 used at 2µg/ml
C: 3T3 cells, ab24700 used at 4µg/ml
D: 3T3 cells, ab24700 used at 6.6µg/ml